Thrombospondin-1 Drives Cardiac Remodeling in Chronic Kidney Disease.

Patients with chronic kidney disease (CKD) face a high risk of cardiovascular disease. Previous studies reported that endogenous thrombospondin 1 (TSP1) involves right ventricular remodeling and dysfunction. Here we show that a murine model of CKD increased myocardial TSP1 expression and produced left ventricular hypertrophy, fibrosis, and dysfunction. TSP1 knockout mice were protected from these features. In vitro, indoxyl sulfate is driving deleterious changes in cardiomyocyte through the TSP1. In patients with CKD, TSP1 and aryl hydrocarbon receptor were both differentially expressed in the myocardium. Our findings summon large clinical studies to confirm the translational role of TSP1 in patients with CKD.

Relation between cardiac magnetic resonance-assessed interstitial fibrosis and diastolic dysfunction in heart failure due to dilated cardiomyopathy.

Background: Dilated cardiomyopathy (DCM) is distinguished by left ventricle (LV) dilation accompanied by systolic dysfunction. However, some studies suggested also a high prevalence of LV diastolic dysfunction (LVDD), similar to a general cohort of heart failure (HF) with reduced ejection fraction (LVEF). The bulk of evidence, mostly arising from basic studies, suggests a causative link between cardiac fibrosis (CF) and LVDD. However, still, there remains a scarcity of data on LVDD and CF. Therefore, the aim of the study was to investigate the association between CF and LVDD in DCM patients. Methods: The study population was composed of 102 DCM patients. Replacement CF was evaluated qualitatively (late gadolinium enhancement - LGE) and quantitively (LGE extent); interstitial cardiac fibrosis was assessed via extracellular volume (ECV). Based on echocardiography patients were divided into normal and elevated left atrial pressure (nLAP, eLAP) groups. Results: 42 % of patients had eLAP. They displayed higher troponin and NT-proBNP. Both groups did not differ in terms of LGE presence and extent; however, eLAP patients had larger ECV: 30.1 ± 5.6 % vs. 27.8 ± 3.9 %, p = 0.03. Moreover, ECV itself was found to be an independent predictor of LVDD (OR = 0.901; 95 %CI 0.810-0.999; p = 0.047; normalised for LVEF and RVOT diameter). Conclusions: More than two-in-five DCM patients had at least moderate LVDD. The mere presence or extent of replacement cardiac fibrosis is similar in patients with nLAP and eLAP. On the other hand, interstitial cardiac fibrosis is more pronounced in those with a higher grade of LVDD. ECV was found to be an independent predictor of LVDD in DCM.

Bone marrow-fibroblast progenitor cell-derived small extracellular vesicles promote cardiac fibrosis via miR-21-5p and integrin subunit αV signalling.

Cardiac fibrosis is the hallmark of cardiovascular disease (CVD), which is leading cause of death worldwide. Previously, we have shown that interleukin-10 (IL10) reduces pressure overload (PO)-induced cardiac fibrosis by inhibiting the recruitment of bone marrow fibroblast progenitor cells (FPCs) to the heart. However, the precise mechanism of FPC involvement in cardiac fibrosis remains unclear. Recently, exosomes and small extracellular vesicles (sEVs) have been linked to CVD progression. Thus, we hypothesized that pro-fibrotic miRNAs enriched in sEV-derived from IL10 KO FPCs promote cardiac fibrosis in pressure-overloaded myocardium. Small EVs were isolated from FPCs cultured media and characterized as per MISEV-2018 guidelines. Small EV's miRNA profiling was performed using Qiagen fibrosis-associated miRNA profiler kit. For functional analysis, sEVs were injected in the heart following TAC surgery. Interestingly, TGFß-treated IL10-KO-FPCs sEV increased profibrotic genes expression in cardiac fibroblasts. The exosomal miRNA profiling identified miR-21a-5p as the key player, and its inhibition with antagomir prevented profibrotic signalling and fibrosis. At mechanistic level, miR-21a-5p binds and stabilizes ITGAV (integrin av) mRNA. Finally, miR-21a-5p-silenced in sEV reduced PO-induced cardiac fibrosis and improved cardiac function. Our study elucidates the mechanism by which inflammatory FPC-derived sEV exacerbate cardiac fibrosis through the miR-21a-5p/ITGAV/Col1α signalling pathway, suggesting miR-21a-5p as a potential therapeutic target for treating hypertrophic cardiac remodelling and heart failure.

Deciphering m6A methylation in monocyte-mediated cardiac fibrosis and monocyte-hitchhiked erythrocyte microvesicle biohybrid therapy.

Rationale: Device implantation frequently triggers cardiac remodeling and fibrosis, with monocyte-driven inflammatory responses precipitating arrhythmias. This study investigates the role of m6A modification enzymes METTL3 and METTL14 in these responses and explores a novel therapeutic strategy targeting these modifications to mitigate cardiac remodeling and fibrosis. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from patients with ventricular septal defects (VSD) who developed conduction blocks post-occluder implantation. The expression of METTL3 and METTL14 in PBMCs was measured. METTL3 and METTL14 deficiencies were induced to evaluate their effect on angiotensin II (Ang II)-induced myocardial inflammation and fibrosis. m6A modifications were analyzed using methylated RNA immunoprecipitation followed by quantitative PCR. NF-κB pathway activity and levels of monocyte migration and fibrogenesis markers (CXCR2 and TGF-ß1) were assessed. An erythrocyte microvesicle-based nanomedicine delivery system was developed to target activated monocytes, utilizing the METTL3 inhibitor STM2457. Cardiac function was evaluated via echocardiography. Results: Significant upregulation of METTL3 and METTL14 was observed in PBMCs from patients with VSD occluder implantation-associated persistent conduction block. Deficiencies in METTL3 and METTL14 significantly reduced Ang II-induced myocardial inflammation and fibrosis by decreasing m6A modification on MyD88 and TGF-ß1 mRNAs. This disruption reduced NF-κB pathway activation, lowered CXCR2 and TGF-ß1 levels, attenuated monocyte migration and fibrogenesis, and alleviated cardiac remodeling. The erythrocyte microvesicle-based nanomedicine delivery system effectively targeted inflamed cardiac tissue, reducing inflammation and fibrosis and improving cardiac function. Conclusion: Inhibiting METTL3 and METTL14 in monocytes disrupts the NF-κB feedback loop, decreases monocyte migration and fibrogenesis, and improves cardiac function. Targeting m6A modifications of monocytes with STM2457, delivered via erythrocyte microvesicles, reduces inflammation and fibrosis, offering a promising therapeutic strategy for cardiac remodeling associated with device implantation.

Apigenin inhibits proliferation and differentiation of cardiac fibroblasts through AKT/GSK3ß signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bunge (S. miltiorrhiza) is an important Traditional Chinese herbal Medicine (TCM) used to treat cardio-cerebrovascular diseases. Based on the pharmacodynamic substance of S. miltiorrhiza, the aim of present study was to investigate the underlying mechanism of S. miltiorrhiza against cardiac fibrosis (CF) through a systematic network pharmacology approach, molecular docking and dynamics simulation as well as experimental investigation in vitro. MATERIALS AND METHODS: A systematic pharmacological analysis was conducted using the Traditional Chinese Medicine Pharmacology (TCMSP) database to screen the effective chemical components of S. miltiorrhiza, then the corresponding potential target genes of the compounds were obtained by the Swiss Target Prediction and TCMSP databases. Meanwhile, GeneCards, DisGeNET, OMIM, and TTD disease databases were used to screen CF targets, and a protein-protein interaction (PPI) network of drug-disease targets was constructed on S. miltiorrhiza/CF targets by Search Tool for the Retrieval of Interacting Genes/Proteins (STING) database. After that, the component-disease-target network was constructed by software Cytoscape 3.7. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed for the intersection targets between drug and disease. The relationship between active ingredient of S. miltiorrhiza and disease targets of CF was assessed via molecular docking and molecular dynamics simulation. Subsequently, the underlying mechanism of the hub compound on CF was experimentally investigated in vitro. RESULTS: 206 corresponding targets to effective chemical components from S. miltiorrhiza were determined, and among them, there were 82 targets that overlapped with targets of CF. Further, through PPI analysis, AKT1 and GSK3ß were the hub targets, and which were both enriched in the PI3K/AKT signaling pathway, it was the sub-pathways of the lipid and atherosclerosis pathway. Subsequently, compound-disease-genes-pathways diagram is constructed, apigenin (APi) was a top ingredients and AKT1 (51) and GSK3ß (22) were the hub genes according to the degree value. The results of molecular docking and dynamics simulation showed that APi has strong affinities with AKT and GSK3ß. The results of cell experiments showed that APi inhibited cells viability, proliferation, proteins expression of α-SMA and collagen I/III, phosphorylation of AKT1 and GSK3ß in MCFs induced by TGFß1. CONCLUSION: Through a systematic network pharmacology approach, molecular docking and dynamics simulation, and confirmed by in vitro cell experiments, these results indicated that APi interacts with AKT and GSK3ß to disrupt the phosphorylation of AKT and GSK3ß, thereby inhibiting the proliferation and differentiation of MCFs induced by TGFß1, which providing new insights into the pharmacological mechanism of S. miltiorrhiza in the treatment of CF.

Effects of sacubitril-valsartan on aging-related cardiac dysfunction.

Heart failure (HF) remains a huge medical burden worldwide, with aging representing a major risk factor. Here, we report the effects of sacubitril/valsartan, an approved drug for HF with reduced EF, in an experimental model of aging-related HF with preserved ejection fraction (HFpEF). Eighteen-month-old female Fisher 344 rats were treated for 12 weeks with sacubitril/valsartan (60 mg/kg/day) or with valsartan (30 mg/kg/day). Three-month-old rats were used as control. No differential action of sacubitril/valsartan versus valsartan alone, either positive or negative, was observed. The positive effects of both sacubitril/valsartan and valsartan on cardiac hypertrophy was evidenced by a significant reduction of wall thickness and myocyte cross-sectional area. Contrarily, myocardial fibrosis in aging heart was not reduced by any treatment. Doppler echocardiography and left ventricular catheterization evidenced diastolic dysfunction in untreated and treated old rats. In aging rats, both classical and non-classical renin-angiotensin-aldosterone system (RAAS) were modulated. In particular, with respect to untreated animals, both sacubitril/valsartan and valsartan showed a partial restoration of cardioprotective non-classical RAAS. In conclusion, this study evidenced the favorable effects, by both treatments, on age-related cardiac hypertrophy. The attenuation of cardiomyocyte size and hypertrophic response may be linked to a shift towards cardioprotective RAAS signaling. However, diastolic dysfunction and cardiac fibrosis persisted despite of treatment and were accompanied by myocardial inflammation, endothelial activation, and oxidative stress.

YAP1-mediated dysregulation of ACE-ACE2 activity augments cardiac fibrosis upon induction of hyperglycemic stress.

BACKGROUND: Diabetic stress acts on the cardiac tissue to induce cardiac hypertrophy and fibrosis. Diabetes induced activated renin angiotensin system (RAS) has been reported to play a critical role in mediating cardiac hypertrophy and fibrosis. Angiotensin converting enzyme (ACE) in producing Angiotensin-II, promotes cardiomyocyte hypertrophy and fibrotic damage. ACE2, a recently discovered molecule structurally homologous to ACE, has been reported to be beneficial in reducing the effect of RAS driven pathologies. METHODS: In vivo diabetic mouse model was used and co-labelling immunostaining assay have been performed to analyse the fibrotic remodeling and involvement of associated target signaling molecules in mouse heart tissue. For in vitro analyses, qPCR and western blot experiments were performed in different groups for RNA and protein expression analyses. RESULTS: Fibrosis markers were observed to be upregulated in the diabetic mouse heart tissue as well as in high glucose treated fibroblast and cardiomyocyte cells. Hyperglycemia induced overexpression of YAP1 leads to increased expression of ß-catenin (CTNNB1) and ACE with downregulated ACE2 expression. The differential expression of ACE/ACE2 promotes TGFB1-SMAD2/3 pathway in the hyperglycemic cardiomyocyte and fibroblast resulting in increased cardiac fibrotic remodeling. CONCLUSION: In the following study, we have reported YAP1 modulates the RAS signaling pathway by inducing ACE and inhibiting ACE2 activity to augment cardiomyocyte hypertrophy and fibrosis in hyperglycemic condition. Furthermore, we have shown that hyperglycemia induced dysregulation of ACE-ACE2 activity by YAP1 promotes cardiac fibrosis through ß-catenin/TGFB1 dependent pathway.

Unlocking the potential of tranilast: Targeting fibrotic signaling pathways for therapeutic benefit.

Fibrosis is the excessive deposition of extracellular matrix in an organ or tissue that results from an impaired tissue repair in response to tissue injury or chronic inflammation. The progressive nature of fibrotic diseases and limited treatment options represent significant healthcare challenges. Despite the substantial progress in understanding the mechanisms of fibrosis, a gap persists translating this knowledge into effective therapeutics. Here, we discuss the critical mediators involved in fibrosis and the role of tranilast as a potential antifibrotic drug to treat fibrotic conditions. Tranilast, an antiallergy drug, is a derivative of tryptophan and has been studied for its role in various fibrotic diseases. These include scleroderma, keloid and hypertrophic scars, liver fibrosis, renal fibrosis, cardiac fibrosis, pulmonary fibrosis, and uterine fibroids. Tranilast exerts antifibrotic effects by suppressing fibrotic pathways, including TGF-ß, and MPAK. Because it disrupts fibrotic pathways and has demonstrated beneficial effects against keloid and hypertrophic scars, tranilast could be used to treat other conditions characterized by fibrosis.

Modulation of anti-cardiac fibrosis immune responses by changing M2 macrophages into M1 macrophages.

BACKGROUND: Macrophages play a crucial role in the development of cardiac fibrosis (CF). Although our previous studies have shown that glycogen metabolism plays an important role in macrophage inflammatory phenotype, the role and mechanism of modifying macrophage phenotype by regulating glycogen metabolism and thereby improving CF have not been reported. METHODS: Here, we took glycogen synthetase kinase 3ß (GSK3ß) as the target and used its inhibitor NaW to enhance macrophage glycogen metabolism, transform M2 phenotype into anti-fibrotic M1 phenotype, inhibit fibroblast activation into myofibroblasts, and ultimately achieve the purpose of CF treatment. RESULTS: NaW increases the pH of macrophage lysosome through transmembrane protein 175 (TMEM175) and caused the release of Ca2+ through the lysosomal Ca2+ channel mucolipin-2 (Mcoln2). At the same time, the released Ca2+ activates TFEB, which promotes glucose uptake by M2 and further enhances glycogen metabolism. NaW transforms the M2 phenotype into the anti-fibrotic M1 phenotype, inhibits fibroblasts from activating myofibroblasts, and ultimately achieves the purpose of treating CF. CONCLUSION: Our data indicate the possibility of modifying macrophage phenotype by regulating macrophage glycogen metabolism, suggesting a potential macrophage-based immunotherapy against CF.

WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts' Activation and Collagen Processing.

Hypertension induces cardiac fibrotic remodelling characterised by the phenotypic switching of cardiac fibroblasts (CFs) and collagen deposition. We tested the hypothesis that Wnt1-inducible signalling pathway protein-1 (WISP-1) promotes CFs' phenotypic switch, type I collagen synthesis, and in vivo fibrotic remodelling. The treatment of human CFs (HCFs, n = 16) with WISP-1 (500 ng/mL) induced a phenotypic switch (α-smooth muscle actin-positive) and type I procollagen cleavage to an intermediate form of collagen (pC-collagen) in conditioned media after 24h, facilitating collagen maturation. WISP-1-induced collagen processing was mediated by Akt phosphorylation via integrin ß1, and disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS-2). WISP-1 wild-type (WISP-1+/+) mice and WISP-1 knockout (WISP-1-/-) mice (n = 5-7) were subcutaneously infused with angiotensin II (AngII, 1000 ng/kg/min) for 28 days. Immunohistochemistry revealed the deletion of WISP-1 attenuated type I collagen deposition in the coronary artery perivascular area compared to WISP-1+/+ mice after a 28-day AngII infusion, and therefore, the deletion of WISP-1 attenuated AngII-induced cardiac fibrosis in vivo. Collectively, our findings demonstrated WISP-1 is a critical mediator in cardiac fibrotic remodelling, by promoting CFs' activation via the integrin ß1-Akt signalling pathway, and induced collagen processing and maturation via ADAMTS-2. Thereby, the modulation of WISP-1 levels could provide potential therapeutic targets in clinical treatment.

Modified mRNA-Mediated CCN5 Gene Transfer Ameliorates Cardiac Dysfunction and Fibrosis without Adverse Structural Remodeling.

Modified mRNAs (modRNAs) are an emerging delivery method for gene therapy. The success of modRNA-based COVID-19 vaccines has demonstrated that modRNA is a safe and effective therapeutic tool. Moreover, modRNA has the potential to treat various human diseases, including cardiac dysfunction. Acute myocardial infarction (MI) is a major cardiac disorder that currently lacks curative treatment options, and MI is commonly accompanied by fibrosis and impaired cardiac function. Our group previously demonstrated that the matricellular protein CCN5 inhibits cardiac fibrosis (CF) and mitigates cardiac dysfunction. However, it remains unclear whether early intervention of CF under stress conditions is beneficial or more detrimental due to potential adverse effects such as left ventricular (LV) rupture. We hypothesized that CCN5 would alleviate the adverse effects of myocardial infarction (MI) through its anti-fibrotic properties under stress conditions. To induce the rapid expression of CCN5, ModRNA-CCN5 was synthesized and administrated directly into the myocardium in a mouse MI model. To evaluate CCN5 activity, we established two independent experimental schemes: (1) preventive intervention and (2) therapeutic intervention. Functional analyses, including echocardiography and magnetic resonance imaging (MRI), along with molecular assays, demonstrated that modRNA-mediated CCN5 gene transfer significantly attenuated cardiac fibrosis and improved cardiac function in both preventive and therapeutic models, without causing left ventricular rupture or any adverse cardiac remodeling. In conclusion, early intervention in CF by ModRNA-CCN5 gene transfer is an efficient and safe therapeutic modality for treating MI-induced heart failure.

Caffeic acid mitigates myocardial fibrosis and improves heart function in post-myocardial infarction by inhibiting transforming growth factor-ß receptor 1 signaling pathways.

Myocardial fibrosis is a pathological, physiological change that results from alterations, such as inflammation and metabolic dysfunction, after myocardial infarction (MI). Excessive fibrosis can cause cardiac dysfunction, ventricular remodeling, and heart failure. Caffeic acid (CA), a natural polyphenolic acid in various foods, has cardioprotective effects. This study aimed to explore whether CA exerts a cardioprotective effect to inhibit myocardial fibrosis post-MI and elucidate the underlying mechanisms. Histological observations indicated that CA ameliorated ventricular remodeling induced by left anterior descending coronary artery ligation in MI mice and partially restored cardiac function. CA selectively targeted transforming growth factor-ß receptor 1 (TGFBR1) and inhibited TGFBR1-Smad2/3 signaling, reducing collagen deposition in the infarcted area of MI mice hearts. Furthermore, cell counting (CCK-8) assay, 5-ethynyl-2'-deoxyuridine assay, and western blotting revealed that CA dose-dependently decreased the proliferation, collagen synthesis, and activation of the TGFBR1-Smad2/3 pathway in primary cardiac fibroblasts (CFs) stimulated by TGF-ß1 in vitro. Notably, TGFBR1 overexpression in CFs partially counteracted the inhibitory effects of CA. These findings suggest that CA effectively mitigates myocardial fibrosis and enhances cardiac function following MI and that this effect may be associated with the direct targeting of TGFBR1 by CA.

Exploring the role of pericardial miRNAs and exosomes in modulating cardiac fibrosis.

The potential of the pericardial space as a therapeutic delivery tool for cardiac fibrosis and heart failure (HF) treatment has yet to be elucidated. Recently, miRNAs and exosomes have been discovered to be present in human pericardial fluid (PF). Novel studies have shown characteristic human PF miRNA compositions associated with cardiac diseases and higher miRNA expressions in PF compared to peripheral blood. Five key studies found differentially expressed miRNAs in HF, angina pectoris, aortic stenosis, ventricular tachycardia, and congenital heart diseases with either atrial fibrillation or sinus rhythm. As miRNA-based therapeutics for cardiac fibrosis and HF showed promising results in several in vivo studies for multiple miRNAs, we hypothesize a potential role of miRNA-based therapeutics delivered through the pericardial cavity. This is underlined by the favorable results of the first phase 1b clinical trial in this emerging field. Presenting the first human miRNA antisense drug trial, inhibition of miR-132 by intravenous administration of a novel antisense oligonucleotide, CDR132L, established efficacy in reducing miR-132 in plasma samples in a dose-dependent manner. We screened the literature, provided an overview of the miRNAs and exosomes present in PF, and drew a connection to those miRNAs previously elucidated in cardiac fibrosis and HF. Further, we speculate about clinical implications and potential delivery methods.

Dapagliflozin reduces systemic inflammation in patients with type 2 diabetes without known heart failure.

OBJECTIVE: Sodium glucose cotransporter 2 (SGLT2) inhibitors significantly improve cardiovascular outcomes in diabetic patients; however, the mechanism is unclear. We hypothesized that dapagliflozin improves cardiac outcomes via beneficial effects on systemic and cardiac inflammation and cardiac fibrosis. RESEARCH AND DESIGN METHODS: This randomized placebo-controlled clinical trial enrolled 62 adult patients (mean age 62, 17% female) with type 2 diabetes (T2D) without known heart failure. Subjects were randomized to 12 months of daily 10 mg dapagliflozin or placebo. For all patients, blood/plasma samples and cardiac magnetic resonance imaging (CMRI) were obtained at time of randomization and at the end of 12 months. Systemic inflammation was assessed by plasma IL-1B, TNFα, IL-6 and ketone levels and PBMC mitochondrial respiration, an emerging marker of sterile inflammation. Global myocardial strain was assessed by feature tracking; cardiac fibrosis was assessed by T1 mapping to calculate extracellular volume fraction (ECV); and cardiac tissue inflammation was assessed by T2 mapping. RESULTS: Between the baseline and 12-month time point, plasma IL-1B was reduced (- 1.8 pg/mL, P = 0.003) while ketones were increased (0.26 mM, P = 0.0001) in patients randomized to dapagliflozin. PBMC maximal oxygen consumption rate (OCR) decreased over the 12-month period in the placebo group but did not change in patients receiving dapagliflozin (- 158.9 pmole/min/106 cells, P = 0.0497 vs. - 5.2 pmole/min/106 cells, P = 0.41), a finding consistent with an anti-inflammatory effect of SGLT2i. Global myocardial strain, ECV and T2 relaxation time did not change in both study groups. GOV REGISTRATION: NCT03782259.

LDHA contributes to nicotine induced cardiac fibrosis through autophagy flux impairment.

Cardiac fibrosis is a typical feature of cardiac pathological remodeling, which is associated with adverse clinical outcomes and has no effective therapy. Nicotine is an important risk factor for cardiac fibrosis, yet its underlying molecular mechanism remains poorly understood. This study aimed to identify its potential molecular mechanism in nicotine-induced cardiac fibrosis. Our results showed nicotine exposure led to the proliferation and transformation of cardiac fibroblasts (CFs) into myofibroblasts (MFs) by impairing autophagy flux. Through the use of drug affinity responsive target stability (DARTS) assay, cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR) technology, it was discovered that nicotine directly increased the stability and protein levels of lactate dehydrogenase A (LDHA) by binding to it. Nicotine treatment impaired autophagy flux by regulating the AMPK/mTOR signaling pathway, impeding the nuclear translocation of transcription factor EB (TFEB), and reducing the activity of cathepsin B (CTSB). In vivo, nicotine treatment exacerbated cardiac fibrosis induced in spontaneously hypertensive rats (SHR) and worsened cardiac function. Interestingly, the absence of LDHA reversed these effects both in vitro and in vivo. Our study identified LDHA as a novel nicotine-binding protein that plays a crucial role in mediating cardiac fibrosis by blocking autophagy flux. The findings suggest that LDHA could potentially serve as a promising target for the treatment of cardiac 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.

Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction.

BACKGROUND: Myocardial infarction (MI) leads to enhanced activity of cardiac fibroblasts (CFs) and abnormal deposition of extracellular matrix proteins, resulting in cardiac fibrosis. Tartrate-resistant acid phosphatase 5 (ACP5) has been shown to promote cell proliferation and phenotypic transition. However, it remains unclear whether ACP5 is involved in the development of cardiac fibrosis after MI. The present study aimed to investigate the role of ACP5 in post-MI fibrosis and its potential underlying mechanisms. METHODS: Clinical blood samples were collected to detect ACP5 concentration. Myocardial fibrosis was induced by ligation of the left anterior descending coronary artery. The ACP5 inhibitor, AubipyOMe, was administered by intraperitoneal injection. Cardiac function and morphological changes were observed on Day 28 after injury. Cardiac CFs from neonatal mice were extracted to elucidate the underlying mechanism in vitro. The expression of ACP5 was silenced by small interfering RNA (siRNA) and overexpressed by adeno-associated viruses to evaluate its effect on CF activation. RESULTS: The expression of ACP5 was increased in patients with MI, mice with MI, and mice with Ang II-induced fibrosis in vitro. AubipyOMe inhibited cardiac fibrosis and improved cardiac function in mice after MI. ACP5 inhibition reduced cell proliferation, migration, and phenotypic changes in CFs in vitro, while adenovirus-mediated ACP5 overexpression had the opposite effect. Mechanistically, the classical profibrotic pathway of glycogen synthase kinase-3ß (GSK3ß)/ß-catenin was changed with ACP5 modulation, which indicated that ACP5 had a positive regulatory effect. Furthermore, the inhibitory effect of ACP5 deficiency on the GSK3ß/ß-catenin pathway was counteracted by an ERK activator, which indicated that ACP5 regulated GSK3ß activity through ERK-mediated phosphorylation, thereby affecting ß-catenin degradation. CONCLUSION: ACP5 may influence the proliferation, migration, and phenotypic transition of CFs, leading to the development of myocardial fibrosis after MI through modulating the ERK/GSK3ß/ß-catenin signaling pathway.

The zinc-finger transcription factor KLF6 regulates cardiac fibrosis.

AIMS: Heart failure (HF) is one of the most devastating consequences of cardiovascular diseases. Regardless of etiology, cardiac fibrosis is present and promotes the loss of heart function in HF patients. Cardiac resident fibroblasts, in response to a host of pro-fibrogenic stimuli, trans-differentiate into myofibroblasts to mediate cardiac fibrosis, the underlying mechanism of which remains incompletely understood. METHODS: Fibroblast-myofibroblast transition was induced in vitro by exposure to transforming growth factor (TGF-ß). Cardiac fibrosis was induced in mice by either transverse aortic constriction (TAC) or by chronic infusion with angiotensin II (Ang II). RESULTS: Through bioinformatic screening, we identified Kruppel-like factor 6 (KLF6) as a transcription factor preferentially up-regulated in cardiac fibroblasts from individuals with non-ischemic cardiomyopathy (NICM) compared to the healthy donors. Further analysis showed that nuclear factor kappa B (NF-κB) bound to the KLF6 promoter and mediated KLF6 trans-activation by pro-fibrogenic stimuli. KLF6 knockdown attenuated whereas KLF6 over-expression enhanced TGF-ß induced fibroblast-myofibroblast transition in vitro. More importantly, myofibroblast-specific KLF6 depletion ameliorated cardiac fibrosis and rescued heart function in mice subjected to the TAC procedure or chronic Ang II infusion. SIGNIFICANCE: In conclusion, our data support a role for KLF6 in cardiac fibrosis.

Oxidative modification of miR-30c promotes cardiac fibroblast proliferation via CDKN2C mismatch.

The response of cardiac fibroblast proliferation to detrimental stimuli is one of the main pathological factors causing heart remodeling. Reactive oxygen species (ROS) mediate the proliferation of cardiac fibroblasts. However, the exact molecular mechanism remains unclear. In vivo, we examined the oxidative modification of miRNAs with miRNA immunoprecipitation with O8G in animal models of cardiac fibrosis induced by Ang II injection or ischemia‒reperfusion injury. Furthermore, in vitro, we constructed oxidation-modified miR-30c and investigated its effects on the proliferation of cardiac fibroblasts. Additionally, luciferase reporter assays were used to identify the target of oxidized miR-30c. We found that miR-30c oxidation was modified by Ang II and PDGF treatment and mediated by excess ROS. We demonstrated that oxidative modification of G to O8G occurred at positions 4 and 5 of the 5' end of miR-30c (4,5-oxo-miR-30c), and this modification promoted cardiac fibroblast proliferation. Furthermore, CDKN2C is a negative regulator of cardiac fibroblast proliferation. 4,5-oxo-miR-30c misrecognizes CDKN2C mRNA, resulting in a reduction in protein expression. Oxidized miR-30c promotes cardiac fibroblast proliferation by mismatch mRNA of CDKN2C.

Macrophage and fibroblast trajectory inference and crosstalk analysis during myocardial infarction using integrated single-cell transcriptomic datasets.

BACKGROUND: Cardiac fibrosis after myocardial infarction (MI) has been considered an important part of cardiac pathological remodeling. Immune cells, especially macrophages, are thought to be involved in the process of fibrosis and constitute a niche with fibroblasts to promote fibrosis. However, the diversity and variability of fibroblasts and macrophages make it difficult to accurately depict interconnections. METHODS: We collected and reanalyzed scRNA-seq and snRNA-seq datasets from 12 different studies. Differentiation trajectories of these subpopulations after MI injury were analyzed by using scVelo, PAGA and Slingshot. We used CellphoneDB and NicheNet to infer fibroblast-macrophage interactions. Tissue immunofluorescence staining and in vitro experiments were used to validate our findings. RESULTS: We discovered two subsets of ECM-producing fibroblasts, reparative cardiac fibroblasts (RCFs) and matrifibrocytes, which appeared at different times after MI and exhibited different transcriptional profiles. We also observed that CTHRC1+ fibroblasts represent an activated fibroblast in chronic disease states. We identified a macrophage subset expressing the genes signature of SAMs conserved in both human and mouse hearts. Meanwhile, the SPP1hi macrophages were predominantly found in the early stages after MI, and cell communication analysis indicated that SPP1hi macrophage-RCFs interactions are mainly involved in collagen deposition and scar formation. CONCLUSIONS: Overall, this study comprehensively analyzed the dynamics of fibroblast and macrophage subsets after MI and identified specific subsets of fibroblasts and macrophages involved in scar formation and collagen deposition.