Neonatal Cardiac Fibroblasts Research Articles

Investigating the role of an orphan nuclear receptor NR4A1 in atrial fibrillation

Abstract Introduction Atrial fibrosis is a major remodelling process in atrial fibrillation (AF). The lack of clinically effective drug targets, due to insufficient understanding of the mechanisms of cardiac fibrosis, hampers the treatment of AF. Orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) is linked to fibrotic disease in multiple organs, however, its role in cardiac fibrosis and AF is yet to be demonstrated. Recent transcriptomic profiling revealed that NR4A1 expression is downregulated in human atrial cardiac fibroblasts (ACFs) in the presence of persistent AF. However, its mechanistic role in atrial fibrogenesis and AF is unknown. Purpose To investigate the expression profile and function of NR4A1 in human ACFs and provide insights into the role of NR4A1 in the context of persistent AF. Methods Human ACFs were isolated (enzymatic digestion) from right and left atrial appendages of 24 patients with persistent AF and controls in sinus rhythm (SR), who had elective heart surgery. NR4A1 knockdown in ACFs was achieved using NR4A1-siRNA. Gene and protein expression were determined by qPCR and western blot respectively, while BrdU assay and scratch assay were used to evaluate cell proliferation and migration. Atrial fibroblasts subpopulations were identified by single-nucleus RNA-sequencing. Subcellular localisation of NR4A1 was assessed by immunostaining, analysed by ImageJ. Results Validation of RNA-sequencing data confirmed a reduction of NR4A1 gene expression in human ACFs in persistent AF by 25% (p=0.029, Fig. 1A) and the NR4A1 predominant abundance in one of three identified subclusters of human ACFs (NR4A1+NAMPT+). The siRNA-mediated knockdown of NR4A1 in ACFs suppressed gene and protein expression of important component of extracellular matrix collagen-1 (Fig. 1B and Fig. 1C) and fibronectin protein (but not mRNA). The NR4A1-deficient cells also had decreased alpha-smooth muscle actin (αSMA) protein and gene expression (Fig. 1D and Fig. 1E), reduced periostin gene expression in the absence of changes in its protein. Furthermore, the NR4A1-deficiency reduced proliferation (by 18%, p=0.005, Fig. 1F) and accelerated migration (p=0.019, Fig. 1G) of human ACFs. These effects were independent of TGF-β1 stimulation, unlike reported in rat neonatal cardiac and dermal fibroblasts. Despite decreased NR4A1 gene expression, patients with AF had doubled NR4A1 protein levels compared to SR-ACFs (p=0.049, Fig. 2A). This was associated with abnormal 1.8-fold increase in the receptor subcellular localisation (p=0.032, Fig. 2B and Fig. 2C), as opposed to the physiological nuclear localisation observed in ACFs in the absence of AF. Conclusion While under physiological conditions, NR4A1 potently activates profibrotic processes in human ACFs, persistent AF patients have increased but abnormally distributed NR4A1 protein in atrial fibroblasts, whose consequences are yet to be determined in order to develop new therapies for AF.Figure 1Figure 2

Direct in vivo cardiac reprogramming with circular RNA of single transcription factor delivered by fibroblast-targeting lipid nanoparticles

Abstract Background Adult mammalian cardiomyocytes (CMs) hold limited capacity for self-regeneration, thus leading to permanent loss of functional myocardium once suffering from injury, especially myocardial infarction (MI). Therefore, cardiac regeneration therapy has been regarded as a potential path to help restore damaged tissue. Direct cardiac reprogramming is one of the most promising ways to boost cardiac regeneration as it could convert resident cardiac fibroblasts into functional induced cardiomyocytes (iCMs). However, difficulty of delivery has been one of the biggest problems that hinder the further clinical translation of this technique such as concerns of mostly used retrovirus vectors and lack of fibroblast-specific targeting ability. In our previous research, we have found that after knocking down of Ybx1 gene, single transcriptional factor Tbx5 could induce cardiac reprogramming of fibroblasts without adding traditional Mef2c and Gata4. But whether this cocktail could achieve in vivo direct cardiac reprogramming still remains to be explored. Objective We planned to develop fibroblast-targeting lipid nanoparticles (LNP) to delivery circular RNA(circRNA) of Tbx5 and small interfering RNA(siRNA) of Ybx1 to realize in vivo cardiac reprogramming in a mouse model of myocardial infarction. Results We first screened different formulations of tdTomato mRNA delivered-LNP both in primary neonatal cardiac fibroblasts and in the myocardial infarction model and found that formulation XF4d could realize specific selectively uptake by cardiac fibroblasts instead of cardiomyocytes. Then we succeeded in synthesizing XF4d LNP delivering Tbx5 circRNA and Ybx1 siRNA (LNPXF4d@Tbx5+siYbx1) and validated its stability at 4°C. Next, after infecting LNPXF4d@Tbx5+siYbx1, primary neonatal CFs and mouse embryonic fibroblasts could be reprogrammed into functional iCMs. We further measured the protein expression of Tbx5 and Ybx1 in CFs after LNPXF4d@Tbx5+siYbx1 treatment. What’s more, we used fibroblast-specific lineage tracing mice (Fsp1Cre/tdTomato mice) to prove the successful conversion of resident CFs into iCMs in the mouse model of myocardial infarction by intramyocardial injection of LNPXF4d@Tbx5+siYbx1. At last, we found that LNPXF4d@Tbx5+siYbx1 could help improve cardiac function after injury and reduce fibrosis, as measured by echocardiography and Masson’s trichrome staining. Conclusion We suggest that our fibroblast specific LNPs delivering single transcriptional factors could a promising method to realize in vivo cardiac reprogramming and help treat myocardial injury, with high possibility of clinical translation and less biosafety concerns.Graphical Abstract

Inhibition of N-acetylglucosaminyltransferase V alleviates diabetic cardiomyopathy in mice by attenuating cardiac hypertrophy and fibrosis

BackgroundThe pathogenesis of diabetic cardiomyopathy is closely linked to abnormal glycosylation modifications. N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the production of N-linked -1–6 branching of oligosaccharides, is involved in several pathophysiological mechanisms of many disorders, including cardiac hypertrophy and heart failure. However, the mechanism by which GnT-V regulates cardiac hypertrophy in diabetic cardiomyopathy is currently poorly understood. In this study, we investigated the role of GnT-V on myocardial hypertrophy in diabetic cardiomyopathy and elucidated the underlying mechanisms.Material and methodsStreptozotocin (STZ) was intraperitoneally injected into mice to induce diabetic cardiomyopathy. An adeno-associated virus (AAV) carrying negative control small hairpin RNA (shNC) or GnT-V-specifc small hairpin RNA (shGnT-V) was used to manipulate GnT-V expression. In our study, forty male C57BL/6J mice were randomly divided into four groups (10 mice per group): control mice with AAV-shNC, diabetic cardiomyopathy mice with AAV-shNC, control mice with AAV-shGnT-V, and diabetic cardiomyopathy mice with AAV-shGnT-V. In addition, H9C2 cells and primary neonatal cardiac fibroblasts treated with high glucose were used as a cell model of diabetes. Analysis of cardiac hypertrophy and fibrosis, as well as functional studies, were used to investigate the underlying molecular pathways.ResultsAAV-mediated GnT-V silencing dramatically improved cardiac function and alleviated myocardial hypertrophy and fibrosis in diabetic mice. In vitro experiments demonstrated that GnT-V was elevated in cardiomyocytes and induced cardiomyocyte hypertrophy in response to high glucose stimulation. GnT-V knockdown significantly reduced the expression of the integrinβ1 signaling pathway, as evidenced by decreased downstream ERK1/2 activity, which inhibited cardiomyocyte hypertrophy accompanied by reduced ANP, BNP, and β-MHC expression. Furthermore, knocking down GnT-V expression lowered the TGF-β1-Smads signaling pathway, which reduced the expression of α-SMA, collagen I, and collagen III.ConclusionsOverall, our research indicated that GnT-V may be a useful therapeutic target to treat diabetic cardiomyopathy, primarily in the inhibition of myocardial hypertrophy and fibrosis.

Acetylcytidine modification of Amotl1 by N-acetyltransferase 10 contributes to cardiac fibrotic expansion in mice after myocardial infarction.

Cardiac fibrosis is a pathological scarring process that impairs cardiac function. N-acetyltransferase 10 (Nat10) is recently identified as the key enzyme for the N4-acetylcytidine (ac4C) modification of mRNAs. In this study, we investigated the role of Nat10 in cardiac fibrosis following myocardial infarction (MI) and the related mechanisms. MI was induced in mice by ligation of the left anterior descending coronary artery; cardiac function was assessed with echocardiography. We showed that both the mRNA and protein expression levels of Nat10 were significantly increased in the infarct zone and border zone 4 weeks post-MI, and the expression of Nat10 in cardiac fibroblasts was significantly higher compared with that in cardiomyocytes after MI. Fibroblast-specific overexpression of Nat10 promoted collagen deposition and induced cardiac systolic dysfunction post-MI in mice. Conversely, fibroblast-specific knockout of Nat10 markedly relieved cardiac function impairment and extracellular matrix remodeling following MI. We then conducted ac4C-RNA binding protein immunoprecipitation-sequencing (RIP-seq) in cardiac fibroblasts transfected with Nat10 siRNA, and revealed that angiomotin-like 1 (Amotl1), an upstream regulator of the Hippo signaling pathway, was the target gene of Nat10. We demonstrated that Nat10-mediated ac4C modification of Amotl1 increased its mRNA stability and translation in neonatal cardiac fibroblasts, thereby increasing the interaction of Amotl1 with yes-associated protein 1 (Yap) and facilitating Yap translocation into the nucleus. Intriguingly, silencing of Amotl1 or Yap, as well as treatment with verteporfin, a selective and potent Yap inhibitor, attenuated the Nat10 overexpression-induced proliferation of cardiac fibroblasts and prevented their differentiation into myofibroblasts in vitro. In conclusion, this study highlights Nat10 as a crucial regulator of myocardial fibrosis following MI injury through ac4C modification of upstream activators within the Hippo/Yap signaling pathway.

Fast but not furious: neonatal cardiac fibroblasts as promoters of regeneration after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Fundação para a Ciência e Tecnologia (FCT) Introduction Mouse neonates at postnatal day 1 (P1) regenerate their hearts after myocardial infarction (MI) but this capacity is transient. Seven day-old animals (P7) develop a reparative response similar to adults, with ischemic tissue being progressively replaced by a rigid fibrotic scar mainly produced by activated cardiac fibroblasts (CF). Recent works support that CF are likely to play an important role in mediating the regeneration-to-repair transition, but the current knowledge is limited. Aims Our final goal is to unravel CF-mediated mechanisms that confer regenerative potential to the neonatal heart and later reactivate these processes in the context of adult MI. Methods Ventricles from non-manipulated mice at different developmental stages were subjected to targeted RNA-seq. CF were isolated by FACS from P1 (regenerative) and P7 (reparative) hearts and characterized at the transcriptional, phenotypic and functional level in vitro. MI was induced by permanent ligation of the left anterior descending coronary artery at P1 and P7. CF and immune cell dynamics were assessed at different timepoints after MI by flow cytometry and immunofluorescence (IF). Transcriptomics of FACS-sorted CF from sham-operated and infarcted hearts at P1 and P7 were assessed by targeted RNA-seq. Intramyocardial injections of either P1 or P7 CF in MI-operated P1 hearts were performed immediately after surgery and hearts were analysed by IF at 7 and 14 days after injury. Results Severe transcriptional alterations on extracellular matrix (ECM) genes were found between P1 (regenerative) and P7 (reparative) hearts. Concomitantly, CF were shown to progressively infiltrate the myocardium and undergo a phenotypic shift that recapitulates transcriptional alterations observed for ECM-encoding genes in the heart. In vitro, CF at P7 show increased proliferation, migration capacity and TGF-β responsiveness. After MI at P1, CF participate in the regenerative response through a fast and transient recruitment to the ischemic site, returning to basal levels at day 7, a period in which CM proliferation and neovascularization are upregulated. Contrarily, MI at P7 resulted in permanent loss of CM, impaired neovascularization and formation of aberrant fibrosis as a result of exuberant and persistent CF recruitment and activation. Whole transcriptomic analysis further showed CF from MI at P7 are highly reactive to injury and engage on the expression of ECM components. The injection of CF resulted in a reduced heart weight and accumulation of cells in the injury site at 7 and 14 days after MI, which suggests a delay in regeneration. Conclusion Physiological CF development after birth entails a shift from a pro-regenerative to a pro-fibrotic phenotype that impacts on the regenerative capacity of the heart and determines the persistent CF activation and permanent scarring after P7.

A transcriptomic analysis of correlation between mitochondrial function and energy metabolism remodeling in mice with myocardial fibrosis following myocardial infarction

To investigate the changes of mitochondrial respiratory function during myocardial fibrosis in mice with myocardial infarction (MI) and its correlation with the increase of glycolytic flux. Forty C57BL/6N mice were randomized into two equal groups to receive sham operation or ligation of the left anterior descending coronary artery to induce acute MI. At 28 days after the operation, 5 mice from each group were euthanized and left ventricular tissue samples were collected for transcriptomic sequencing. FPKM method was used to calculate gene expression levels to identify the differentially expressed genes (DEGs) in MI mice, which were analyzed using GO and KEGG databases to determine the pathways affecting the disease process. Heat maps were drawn to show the differential expressions of the pathways and the related genes in the enrichment analysis. In primary cultures of neonatal mouse cardiac fibroblasts (CFs), the changes in mitochondrial respiration and glycolysis levels in response to treatment with the pro-fibrotic agonist TGF-β1 were analyzed using Seahorse experiment. The mouse models of MI showed significantly increased diastolic and systolic left ventricular diameter (P < 0.05) and decreased left ventricular ejection fraction (P < 0.0001). A total of 124 up-regulated and 106 down-regulated DEGs were identified in the myocardial tissues of MI mice, and GO and KEGG enrichment analysis showed that these DEGs were significantly enriched in fatty acid metabolism, organelles and other metabolic pathways and in the mitochondria. Heat maps revealed fatty acid beta oxidation, mitochondrial dysfunction and increased glycolysis levels in MI mice. In the primary culture of CFs, treatment with TGF-β1 significantly reduced the basal and maximum respiratory levels and increased the basal and maximum glycolysis levels (P < 0.0001). During myocardial fibrosis, energy metabolism remodeling occurs in the CFs, manifested by lowered mitochondrial function and increased energy generation through glycolysis.

BAG3 localizes to mitochondria in cardiac fibroblasts and regulates mitophagy.

The co-chaperone Bcl2-associated athanogene 3 (BAG3) is a central node in protein quality control in the heart. In humans and animal models, decreased BAG3 expression is associated with cardiac dysfunction and dilated cardiomyopathy. Although previous studies focused on BAG3 in cardiomyocytes, cardiac fibroblasts are also critical drivers of pathologic remodeling. Yet, the role of BAG3 in cardiac fibroblasts is almost completely unexplored. Here, we show that BAG3 is expressed in primary rat neonatal cardiac fibroblasts and preferentially localizes to mitochondria. Knockdown of BAG3 reduces mitophagy and enhances fibroblast activation, which is associated with fibrotic remodeling. Heat shock protein 70 (Hsp70) is a critical binding partner for BAG3 and inhibiting this interaction in fibroblasts using the drug JG-98 decreased autophagy, decreased mitofusin-2 expression, and disrupted mitochondrial morphology. Together, these data indicate that BAG3 is expressed in cardiac fibroblasts, where it facilitates mitophagy and promotes fibroblast quiescence. This suggests that depressed BAG3 levels in heart failure may exacerbate fibrotic pathology, thus contributing to myocardial dysfunction through sarcomere-independent pathways.NEW & NOTEWORTHY We report BAG3's localization to mitochondria and its role in mitophagy for the first time in primary ventricular cardiac fibroblasts. We have also collected the first evidence showing that loss of BAG3 increases cardiac fibroblast activation into myofibroblasts, which are major drivers of cardiac fibrosis and pathological remodeling during heart disease.

TAX1BP1 downregulation by STAT3 in cardiac fibroblasts contributes to diabetes-induced heart failure with preserved ejection fraction

Heart failure (HF) with preserved ejection fraction (HFpEF) is now the most common form of HF and has been reported to be closely related to diabetes. Accumulating evidence suggests that HFpEF patients exhibit cardiac fibrosis. This study investigates whether direct targeted inhibition of the activation of cardiac fibroblasts (CFs), the main effector cells in cardiac fibrosis, improves diabetes-induced HFpEF and elucidates the underlying mechanisms. Twenty-week-old db/db mice exhibited HFpEF, as confirmed by echocardiography and hemodynamic measurements. Proteomics was performed on CFs isolated from the hearts of 20-week-old C57BL/6 and db/db mice. Bioinformatic prediction was used to identify target proteins. Experimental validation was performed in both high glucose (HG)-treated neonatal mouse CFs (NMCFs) and diabetic hearts. TAX1 binding protein 1 (TAX1BP1) was identified as the most significantly differentially expressed protein between 20-week-old C57BL/6 and db/db mice. TAX1BP1 mRNA and protein were markedly downregulated in CFs from diabetic hearts and HG-cultured NMCFs. Overexpression of TAX1BP1 profoundly inhibited HG/diabetes-induced NF-κB nuclear translocation and collagen synthesis in CFs, improved cardiac fibrosis, hypertrophy, inflammation and HFpEF in diabetic mice. Mechanistically, signal transducer and activator of transcription 3 (STAT3), which is phosphorylated and translocated from the cytoplasm into the nucleus under hyperglycemic conditions, bound to TAX1BP1 promoter and blocked TAX1BP1 transcriptional activity, consequently promoting NF-κB nuclear translocation and collagen synthesis in CFs, aggravating cardiac fibrosis, hypertrophy and inflammation, leading to HFpEF in db/db mice. Taken together, our findings demonstrate that targeting regulation of STAT3-TAX1BP1-NF-κB signaling in CFs may be a promising therapeutic approach for diabetes-induced HFpEF.

Cardiac fibroblasts-mtExosomes-macrophages axis aggravates ventricular remodeling after acute myocardial infarction

Abstract Background Intercellular communication between cardiac fibroblasts (CFs) and macrophages plays an important role in the progression of ventricular remodeling after acute myocardial infarction (AMI). Following intense oxidative stress, activation of mitochondrial quality control (MQC) systems is necessary to eliminate damaged components. Recent studies have highlighted the emergence of a new form of MQC called mitochondrial-derived vesicles (MDVs), which can be released into the extracellular space via exosomes. Our previous research has revealed that exosomes derived from fibroblasts treated with oxygen-glucose deprivation (OGD) contained mitochondrial damage components, which we named "mtExosomes". The uptake of mtExosomes by macrophages can significantly promote inflammatory response. Purpose To explore the specific effects of CFs-mtExosomes-macrophages communication axis on ventricular remodeling after AMI. Methods Male C57 mice underwent left anterior descending coronary artery-ligation surgery for orthotopic myocardial injection of CFs derived exosomes. Primary neonatal mouse CFs were isolated and cultured for 24h in sugar-free medium with 1% O2 (OGD) to mimic ischemic injury in vitro. Conditioned medium was separated to obtain exosomes by ultracentrifugation. Label-free quantitative proteomics was used to identify differentially expressed proteins in exosomes. The damaged mitochondrial components in the exosomes were identified using mito-tracker and nanoflowmetry. After co-culture with OGD-CFs derived exosomes for 24h, RNA-seq was performed to analyze the activated inflammatory signaling pathways in bone marrow-derived macrophages. Results OGD-CFs derived exosomes significantly reduced cardiac function post-surgery day 3 in model mice, and fibrotic remodeling was observed at 28 days. In vitro co-culture experiments revealed that the conditioned medium from OGD-CFs increased the expression and secretion of IL-1β and IL-6 in macrophages. Separation of secretory components by differential centrifugation suggested that exosomes had the most significant pro-inflammatory effect on macrophages. Exosome proteomics showed that fibroblast-derived exosomes were rich in mitochondrial proteins. A significant fluorescence signal was detected by nanoflowmetry after incubation of OGD-CFs-derived exosomes with Mito-tracker. OGD-CFs derived exosomes also had higher mtDNA, mtROS, and ATP levels. RNA-seq enrichment analysis revealed significant activation of Toll-like receptor and NOD-like receptor signaling pathways in macrophages. Western blot analysis further confirmed the up-regulation of NLRP3/Caspase-1/IL-1β and TLR9/NF-κB/IL-6 signaling pathways. Conclusion Following ischemic injury, CFs in infarction area release exosomes rich in damaged mitochondria (mtExosomes). The uptake of mtExosomes by macrophages promotes the secretion of IL-6 and IL-1β via NLRP3/Caspase-1/IL-1β and TLR9/NF-κB/IL-6 signaling pathways, leading to ventricular remodeling after AMI.Graphical Abstract

Protein tyrosine phosphatase sigma and zeta1 regulate cardiac fibroblast contractile function through TGF-beta dependent and independent pathways

Abstract Background Heart failure is a frequently fatal cardiovascular disease with limited therapeutic options. To improve our understanding of signaling pathways in affected cells is therefore important. Protein tyrosine phosphatase sigma (PTPRσ) and zeta 1 (PTPRz1) have been shown to be involved in cardiac development and disease [1,2]. Both phosphatases are upregulated in patients with end stage heart failure. We demonstrated that PTPRz1 inhibits while PTPRσ is required for migration of neonatal cardiac fibroblasts (nCF) [3]. Purpose To further evaluate the influence of PTPRσ and PTPRz1 on fibroblast phenotype with regard to stress fiber formation and collagen contraction and their interaction with the TGFβ pathway. Methods nCF were transfected with either scrambled-, PTPRσ-, PTPRz1-, or both siRNAs. mRNA lysates were harvested 24h after transfection with subsequent rt-PCR analysis for the phosphatases. Transfected cells were seeded and stained with labeled phalloidin to measure the formation of stress fibers or in collagen gel matrices to evaluate their contractile capacities. To evaluate the nuclear translocation of SMAD3 immunohistochemical colocalization of SMAD3 and DAPI was quantified. Data were analyzed by Kruskal-Wallis and Mann-Whitney-U or ANOVA followed by t-test with Welsh correction using GraphPad prism. Results PTPRz1 and -σ knockdown (kd) was highly efficient (&amp;gt;85%). PTPRz1 mRNA was upregulated by 48% after PTPRσ kd (p &amp;lt; 0.05, n=4) but not vice versa and not after combined kd. Compared to control, nCF showed a 26% increase in the number of stress fibers in response to the PTPRz1 kd (p = 0.057, n=3-4) and an increase of 29% after PTPRσ/-z1 kd (p &amp;lt; 0.05, n=4). Collagen gels were contracted to about 70% of their surface by TGFβ in control (p&amp;lt;0.05, n=4) or any kd condition. They were also contracted by 24,5% after PTPRz1 knockdown (p &amp;lt; 0.01, n=4), whereas the additional kd of PTPRσ obliterated the effect. Nuclear translocation of SMAD3 was roughly doubled upon TGFβ stimulation in controls and all kd conditions. In unstimulated nCF, PTPRσ kd induced a 28%, PTPRz1 kd a 45%, and their combination a 114% increase of nuclei positive for SMAD3 (p &amp;lt; 0.05). Conclusion SMAD3 nuclear translocation is additively induced by PTPRz1 and PTPRσ kd independent of TGFβ. Stress fiber formation, however, seems to be inhibited by PTPRz1 independent of PTPRσ, while PTPRz1-kd-induced collagen contraction does depend on the presence of PTPRσ. PTPRσ and PTPRz1 thus have separate, overlapping and antagonistic effects that are TGFβ dependent and independent. The elucidation of the involvement of the phosphatases in fibroblast signaling and function could improve our understanding of adverse remodeling during heart failure and deliver novel options for therapeutic interventions.

Exogenous Spermidine Alleviates Diabetic Myocardial Fibrosis Via Suppressing Inflammation and Pyroptosis in db/db Mice

The main pathological feature of diabetic cardiomyopathy (DCM) caused by diabetes mellitus is myocardial fibrosis. According to recent studies in cardiology, it has been suggested that spermidine (SPD) has cardioprotective properties. To explore the role and mechanism of SPD in alleviating myocardial fibrosis of DCM. In vivo and in vitro study. Type 2 diabetic mice and primary neonatal mouse cardiac fibroblasts (CFs) were selected. Measurements of serum-related markers, echocardiographic analysis, and immunohistochemistry were used to evaluate myocardial fibrosis injury and the effects of SPD. The proliferation and migration of CFs undergoing different treatments were studied. Immunoblotting and real-time quantitative reverse transcription polymerase chain reaction were used to demonstrate molecular mechanisms. In vivo immunoblotting analysis indicated a downregulation of ornithine decarboxylase and an upregulation of SPD/spermine N1-acetyltransferase. We observed cardiac dysfunction in diabetic mice after 12 weeks. However, the administration of exogenous SPD improved cardiac function, decreased collagen deposition, and reduced myocardial tissue damage. mRNA expression levels of NLRP3, Caspase-1, GSDMD-N, interleukin (IL)-1β, IL-17A, and IL-18 were increased and suppressed in the myocardium of db/db mice upon treatment with SPD. SPD inhibited the proliferation, migration, and collagen secretion of high-glucose-treated fibroblasts in vitro. SPD inhibits the activation of the TGF-β1/Smad signaling pathway and decreases collagen deposition by reducing pyroptosis and Smad-7 ubiquitination levels. Based on our findings, SPD may have potential applications in protecting against the deterioration of cardiac function in patients with DCM due to a significant new mechanism for diabetic myocardial fibrosis that we discovered.

Abstract P1068: Yap Induces A Neonatal Like Pro-renewal Niche In The Adult Heart

The heart is a poorly renewable organ, meaning that tissue architecture and the cardiac microenvironment are irreversibly disrupted after injury. This presents a major challenge in treatment of ischemic heart disease, the leading cause of death worldwide. To date, most studies have focused on cardiomyocytes (CMs). However, very few studies have examined CM-extrinsic mechanisms, such as innate immunity, that complement CM-intrinsic mechanisms in heart renewal. Inactivation of the Hippo signaling pathway can rebuild the post ischemic microenvironment and improve cardiac function. We investigated spatially resolved cellular relationships of neonatal and adult renewal-competent hearts to gain insight into inefficient mammalian heart renewal. Spatial transcriptomics and single-cell sequencing of adult control hearts and hearts expressing YAP5SA, an active version of the Hippo signaling pathway effector YAP, which models heart renewal, revealed a conserved, renewal-competent CM population in control hearts and amplified in YAP5SA hearts. This CM population was also found in the wildtype, renewal competent neonatal heart after myocardial infarction, as well as the adult human heart. In YAP5SA hearts and neonatal hearts post myocardial infarction, resident macrophages and cardiac fibroblasts expressing complement pathway genes colocalized with these CMs, creating a pro-renewal microenvironment. Complement loss-of-function in fibroblasts or macrophages suppressed cardiomyocyte proliferation in both neonatal injured hearts and adult YAP5SA hearts, indicating that the complement system plays a direct role in heart renewal.

Cytokine receptor-like factor 1 (CRLF1) promotes cardiac fibrosis via ERK1/2 signaling pathway.

Cardiac fibrosis is a cause of morbidity and mortality in people with heart disease. Anti-fibrosis treatment is a significant therapy for heart disease, but there is still no thorough understanding of fibrotic mechanisms. This study was carried out to ascertain the functions of cytokine receptor-like factor 1 (CRLF1) in cardiac fibrosis and clarify its regulatory mechanisms. We found that CRLF1 was expressed predominantly in cardiac fibroblasts. Its expression was up-regulated not only in a mouse heart fibrotic model induced by myocardial infarction, but also in mouse and human cardiac fibroblasts provoked by transforming growth factor-‍β1 (TGF‍-‍β1). Gain- and loss-of-function experiments of CRLF1 were carried out in neonatal mice cardiac fibroblasts (NMCFs) with or without TGF-‍β1 stimulation. CRLF1 overexpression increased cell viability, collagen production, cell proliferation capacity, and myofibroblast transformation of NMCFs with or without TGF‍-‍β1 stimulation, while silencing of CRLF1 had the opposite effects. An inhibitor of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and different inhibitors of TGF-‍β1 signaling cascades, comprising mothers against decapentaplegic homolog (SMAD)‍-dependent and SMAD-independent pathways, were applied to investigate the mechanisms involved. CRLF1 exerted its functions by activating the ERK1/2 signaling pathway. Furthermore, the SMAD-dependent pathway, not the SMAD-independent pathway, was responsible for CRLF1 up-regulation in NMCFs treated with TGF-‍β1. In summary, activation of the TGF-‍β1/SMAD signaling pathway in cardiac fibrosis increased CRLF1 expression. CRLF1 then aggravated cardiac fibrosis by activating the ERK1/2 signaling pathway. CRLF1 could become a novel potential target for intervention and remedy of cardiac fibrosis.

EphrinB2 drives osteogenic fate of adult cardiac fibroblasts in a calcium influx dependent manner.

Cardiac calcification is a crucial but underrecognized pathological process, greatly increasing the risk of cardiovascular diseases. Little is known about how cardiac fibroblasts, as a central mediator, facilitate abnormal mineralization. Erythropoietin-producing hepatoma interactor B2 (EphrinB2), previously identified as an angiogenic regulator, is involved in fibroblast activation, while its role in the osteogenic differentiation of cardiac fibroblasts is unknown. Bioinformatics analysis was conducted to characterize the expression of the Ephrin family in human calcified aortic valves and calcific mouse hearts. The effects of EphrinB2 on cardiac fibroblasts to adopt osteogenic fate was determined by gain- and loss-of-function. EphrinB2 mRNA level was downregulated in calcified aortic valves and mouse hearts. Knockdown of EphrinB2 attenuated mineral deposits in adult cardiac fibroblasts, whereas overexpression of EphrinB2 promoted their osteogenic differentiation. RNA sequencing data implied that Ca2+-related S100/receptor for advanced glycation end products (RAGE) signaling may mediate EphrinB2-induced mineralization in cardiac fibroblasts. Moreover, L-type calcium channel blockers inhibited osteogenic differentiation of cardiac fibroblasts, implying a critical role in Ca2+ influx. In conclusion, our data illustrated an unrecognized role of EphrinB2, which functions as a novel osteogenic regulator in the heart through Ca2+ signaling and could be a potential therapeutic target in cardiovascular calcification.NEW & NOTEWORTHY In this study, we observed that adult cardiac fibroblasts but not neonatal cardiac fibroblasts exhibit the ability of osteogenic differentiation. EphrinB2 promoted osteogenic differentiation of cardiac fibroblasts through activating Ca2+-related S100/RAGE signaling. Inhibition of Ca2+ influx using L-type calcium channel blockers inhibited EphrinB2-mediated calcification of cardiac fibroblasts. Our data implied an unrecognized role of EphrinB2 in regulating cardiac calcification though Ca2+-related signaling, suggesting a potential therapeutic target of cardiovascular calcification.

The p53/miR-29a-3p axis mediates the antifibrotic effect of leonurine on angiotensin II-stimulated rat cardiac fibroblasts.

Overactivation of cardiac fibroblasts (CFs) is one of the main causes of myocardial fibrosis (MF), and inhibition of CF activation is a crucial strategy for MF therapy. A previous study by our group demonstrated that leonurine (LE) effectively inhibits collagen synthesis and myofibroblast generation originated from CFs, and eventually mitigates the progression of MF (where miR-29a-3p is likely to be a vital mediator). However, the underlying mechanisms involved in this process remain unknown. Thus, the present study aimed to investigate the precise role of miR-29a-3p in LE-treated CFs, and to elucidate the pharmacological effects of LE on MF. Neonatal rat CFs were isolated and stimulated by angiotensin II (Ang II) to mimic the pathological process of MF in vitro. The results show that LE distinctly inhibits collagen synthesis, as well as the proliferation, differentiation and migration of CFs, all of which could be induced by Ang II. In addition, LE promotes apoptosis in CFs under Ang II stimulation. During this process, the down-regulated expressions of miR-29a-3p and p53 are partly restored by LE. Either knockdown of miR-29a-3p or inhibition of p53 by PFT-α (a p53 inhibitor) blocks the antifibrotic effect of LE. Notably, PFT-α suppresses miR-29a-3p levels in CFs under both normal and Ang II-treated conditions. Furthermore, ChIP analysis confirmed that p53 is bound to the promoter region of miR-29a-3p, and directly regulates its expression. Overall, our study demonstrates that LE upregulates p53 and miR-29a-3p expression, and subsequently inhibits CF overactivation, suggesting that the p53/miR-29a-3p axis may play a crucial role in mediating the antifibrotic effect of LE against MF.

Neonatal and adult cardiac fibroblasts exhibit inherent differences in cardiac regenerative capacity

Directly reprogramming fibroblasts into cardiomyocytes improves cardiac function in the infarcted heart. However, the low efficacy of this approach hinders clinical applications. Unlike the adult mammalian heart, the neonatal heart has an intrinsic regenerative capacity. Consequently, we hypothesized that birth imposes fundamental changes in cardiac fibroblasts which limit their regenerative capabilities. In support, we found that reprogramming efficacy invitro was markedly lower with fibroblasts derived from adult mice versus those derived from neonatal mice. Notably, fibroblasts derived from adult mice expressed significantly higher levels of pro-angiogenic genes. Moreover, under conditions that promote angiogenesis, only fibroblasts derived from adult mice differentiated into tube-like structures. Targeted knockdown screening studies suggested a possible role for the transcription factor Epas1. Epas1 expression was higher in fibroblasts derived from adult mice, and Epas1 knockdown improved reprogramming efficacy in cultured adult cardiac fibroblasts. Promoter activity assays indicated that Epas1 functions as both a transcription repressor and an activator, inhibiting cardiomyocyte genes while activating angiogenic genes. Finally, the addition of an Epas1 targeting siRNA to the reprogramming cocktail markedly improved reprogramming efficacy invivo with both the number of reprogramming events and cardiac function being markedly improved. Collectively, our results highlight differences between neonatal and adult cardiac fibroblasts and the dual transcriptional activities of Epas1 related to reprogramming efficacy.

Shexiang Tongxin Dropping Pill Allieviates Heart Failure via Extracellula Matrix-Receptor Interaction Pathways Based on RNA-Seq Transcriptomics and Experimental Studies.

To investigate the protective mechanisms of Chinese medicine Shexiang Tongxin Dropping Pills (STDP) on heart failure (HF). Isoproterenol (ISO)-induced HF rat model and angiotensin II (Ang II)-induced neonatal rat cardiac fibroblast (CFs) model were used in the present study. HF rats were treated with and without STDP (3 g/kg). RNA-seq was performed to identify differentially expressed genes (DEGs). Cardiac function was evaluated by echocardiography. Hematoxylin and eosin and Masson's stainings were taken to assess cardiac fibrosis. The levels of collagen I (Col I) and collagen III (Col III) were detected by immunohistochemical staining. CCK8 kit and transwell assay were implemented to test the CFs' proliferative and migratory activity, respectively. The protein expressions of α-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), MMP-9, Col I, and Col III were detected by Western blotting. The results of RNA-seq analysis showed that STDP exerted its pharmacological effects on HF via multiple signaling pathways, such as the extracellular matrix (ECM)-receptor interaction, cell cycle, and B cell receptor interaction. Results from in vivo experiments demonstrated that STDP treatment reversed declines in cardiac function, inhibiting myocardial fibrosis, and reversing increases in Col I and Col III expression levels in the hearts of HF rats. Moreover, STDP (6, 9 mg/mL) inhibited the proliferation and migration of CFs exposed to Ang II in vitro (P<0.05). The activation of collagen synthesis and myofibroblast generation were markedly suppressed by STDP, also the synthesis of MMP-2 and MMP-9, as well as ECM components Col I, Col III, and α-SMA were decreased in Ang II-induced neonatal rats' CFs. STDP had anti-fibrotic effects in HF, which might be caused by the modulation of ECM-receptor interaction pathways. Through the management of cardiac fibrosis, STDP may be a compelling candidate for improving prognosis of HF.

RIP2 inhibition alleviates lipopolysaccharide-induced septic cardiomyopathy via regulating TAK1 signaling

PurposeRIP2 is a member of the receptor-interacting protein family that has been associated with various pathophysiological processes, including immunity, apoptosis, and autophagy. However, no studies have hitherto reported the role of RIP2 in lipopolysaccharide (LPS)-induced septic cardiomyopathy (SCM). This study was designed to illustrate the role of RIP2 in LPS-induced SCM. MethodsC57 and RIP2 knockout mice received intraperitoneal injections of LPS to establish models of SCM. Echocardiography was used to assess the cardiac function of the mice. Real-time-PCR, cytometric bead array and immunohistochemical staining were used to detect the inflammatory response. Immunoblotting was used to determine the protein expression of relevant signaling pathways. Our findings were validated by treatment with a RIP2 inhibitor. Neonatal rats cardiomyocytes (NRCMs) and cardiac fibroblasts (CFs) were transfected with Ad-RIP2 to further explore the role of RIP2 in vitro. ResultsRIP2 expression was upregulated in our mice models of septic cardiomyopathy and LPS-stimulated cardiomyocytes and fibroblasts. RIP2 knockout or RIP2 inhibitors attenuated LPS-induced cardiac dysfunction and reduced the inflammatory response in mice. Overexpression of RIP2 in vitro enhanced the inflammatory response, and TAK1 inhibitors attenuated the inflammatory response caused by overexpression of RIP2. ConclusionOur findings substantiate that RIP2 induces an inflammatory response by regulating the TAK1/IκBα/NF-κB signaling pathway. RIP2 inhibition by genetic or pharmacological approaches has huge prospects for application as a potential treatment strategy for inhibiting inflammation, alleviating cardiac dysfunction, and improving survival.

Gβγ subunits colocalize with RNA polymerase II and regulate transcription in cardiac fibroblasts

Gβγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gβγ signaling, we investigated the functional role of Gβγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gβ1γ dimer in the fibrotic response. Depletion of Gβ1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gβ1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gβγ with transcription complexes. Together, our findings suggest that Gβ1γ is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gβγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gβγ signaling in other cell types.

Polypyrimidine tract binding protein 1 exacerbates cardiac fibrosis by regulating fatty acid-binding protein 5.

The activation of cardiac fibroblasts (CFs) leads to overproduction of collagens and subsequently cardiac fibrosis. However, the regulatory mechanism of CF function in the process of cardiac fibrosis remains unclear. This work investigated the function of polypyrimidine tract binding protein 1 (PTBP1)/nuclear receptor NR4A1 (Nur77)/fatty acid-binding protein 5 (FABP5) axis in myocardial fibrosis. Cardiac fibrosis was induced in mice suffered left anterior descending ligation. In parallel, neonatal mouse CFs were isolated and stimulated with transforming growth factor-β1 (TGF-β1). Cardiac fibrosis was evaluated by Masson's trichrome staining. Expression of PTBP1, Nur77, FABP5, collagen I, and collagen III was measured by quantitative real-time PCR and western blotting. Proliferation of CFs was assessed by 5-ethynyl-2'-deoxyuridine assay. Molecular interaction was validated by RNA-binding protein immunoprecipitation, chromatin immunoprecipitation, and dual luciferase reporter assay. PTBP1 was up-regulated (P<0.05), whereas Nur77 (P<0.05) and FABP5 (P<0.05) were down-regulated in the fibrotic hearts of mice and TGF-β1-exposed CFs. PTBP1 overexpression facilitated proliferation (P<0.05) and collagen I (P<0.05) and collagen III (P<0.05) expression of CFs after stimulation with TGF-β1. PTBP1 reduced Nur77 stability (P<0.05) to inhibit Nur77 expression (P<0.05) in CFs. Nur77 bound to FABP5 promoter to promote the transcription (P<0.05) and expression (P<0.05) of FABP5. Silencing of Nur77 or FABP5 abolished the inhibitory effect of PTBP1 knockdown on proliferation (P<0.05) and collagen I (P<0.05) and collagen III (P<0.05) expression of CFs in vitro. PTBP1 depletion ameliorated cardiac fibrosis (P<0.05), α-smooth muscle actin (P<0.05), and collagen I (P<0.05) expression in myocardial infarction mice through regulating Nur77/FABP5 pathway (P<0.05) in vivo. PTBP1 contributed to cardiac fibrosis via promoting CF proliferation and collagen deposition through Nur77 mRNA decay and subsequent transcription inhibition of FABP5. Our findings suggest that PTBP1/Nur77/FABP5 axis may be potential targets for cardiac fibrosis therapy.