Response Of Cardiac Fibroblasts Research Articles

Mechanical stimulation of induced pluripotent stem derived cardiac fibroblasts

Cardiac fibrosis contributes to the development of heart failure, and is the response of cardiac fibroblasts (CFs) to pressure or volume overload. Limiting factors in CFs research are the poor availability of human cells and the tendency of CFs to transdifferentiate into myofibroblasts when cultured in vitro. The possibility to generate CFs from induced pluripotent stem cells (iPSC), providing a nearly unlimited cell source, opens new possibilities. However, the behaviour of iPSC-CFs under mechanical stimulation has not been studied yet. Our study aimed to assess the behaviour of iPSC-CFs under mechanical stretch and pro-fibrotic conditions. First, we confirm that iPSC-CFs are comparable to primary CFs at gene, protein and functional level. Furthermore, iPSC-derived CFs adopt a pro-fibrotic response to transforming growth factor beta (TGF-β). In addition, mechanical stretch inhibits TGF-β-induced fibroblast activation in iPSC-CFs. Thus, the responsiveness to cytokines and mechanical stimulation of iPSC-CFs demonstrates they possess key characteristics of primary CFs and may be useful for disease modelling.

A Dynamic Gradient Stiffness Material Platform to Manipulate Cardiac Fibroblasts' Spatio‐Temporal Behavior

AbstractAfter myocardial infarction, there exists a spatiotemporal variation of cardiac tissue stiffness across the infarcted border region outward to remote regions, influencing adverse remodeling and cardiac fibrosis, and this stiffness gradient changes over time. Here, a platform with dynamic, tunable, and reversible gradient stiffness can recapitulate in vitro the time‐dependent stiffness range across the infarction border that occurs as part of the remodeling process is presented. This platform enables the observation of time‐dependent interaction between cardiac fibroblasts and their mechanical microenvironment in a spatiotemporal manner. Specifically, the competition and cooperation of a chemical cue (antifibrotic drug) and mechanical cue (gradient softening) in tandem to attenuate the fibrotic responses of cardiac fibroblasts is examined. Applying a combined intervention showed either additive or antagonistic effects on fibrosis‐related gene regulation compared to separate interventions of drug or softening. This work reveals the spatiotemporal variation of fibrotic response in cardiac fibroblasts as well as the complexity of antifibrotic drug dosing with stiffness changes and their combinatory effect on cardiac fibroblasts. This platform provides a unique in vitro tool to study disease progression mechanisms in a more clinically relevant microenvironment and also serves as a cost‐effective model for potential therapeutic screening.

Fibroblasts under pressure: cardiac fibroblast responses to hypertension and antihypertensive therapies.

Approximately 50% of Americans have hypertension, which significantly increases the risk of heart failure. In response to increased peripheral resistance in hypertension, intensified mechanical stretch in the myocardium induces cardiomyocyte hypertrophy and fibroblast activation to withstand increased pressure overload. This changes the structure and function of the heart, leading to pathological cardiac remodeling and eventual progression to heart failure. In the presence of hypertensive stimuli, cardiac fibroblasts activate and differentiate to myofibroblast phenotype capable of enhanced extracellular matrix secretion in coordination with other cell types, mainly cardiomyocytes. Both systemic and local renin-angiotensin-aldosterone system activation lead to increased angiotensin II stimulation of fibroblasts. Angiotensin II directly activates fibrotic signaling such as transforming growth factor β/SMAD and mitogen-activated protein kinase (MAPK) signaling to produce extracellular matrix comprised of collagens and matricellular proteins. With the advent of single-cell RNA sequencing techniques, heterogeneity in fibroblast populations has been identified in the left ventricle in models of hypertension and pressure overload. The various clusters of fibroblasts reveal a range of phenotypes and activation states. Select antihypertensive therapies have been shown to be effective in limiting fibrosis, with some having direct actions on cardiac fibroblasts. The present review focuses on the fibroblast-specific changes that occur in response to hypertension and pressure overload, the knowledge gained from single-cell analyses, and the effect of antihypertensive therapies. Understanding the dynamics of hypertensive fibroblast populations and their similarities and differences by sex is crucial for the advent of new targets and personalized medicine.

Single cell RNA sequencing to dissect transcriptional signature of cardiac fibroblasts activation in experimental autoimmune myocarditis

Abstract Introduction Dilated cardiomyopathy (DCM) is a condition that evolves from inflammation of the myocardium, and it is often associated with a poor prognosis and fatal outcome. Experimental autoimmune myocarditis (EAM) represents a CD4+ T cell-dependent animal model of acute myocarditis followed by the development of post-inflammatory DCM. Purpose The aim of the study was to investigate the transcriptional changes in cardiac fibroblasts during disease progression in a mouse model of EAM at the single cell level. Methods 6–8-week-old BALB/c mice (or reporter mice expressing EGFP under the collagen type I promoter) were immunized with αMyHC peptide together with Complete Freunds’ Adjuvant. Live cardiac cells were sorted from healthy hearts (d0), at acute myocarditis (d19), during resolution of inflammation (d25), and at the DCM stage (d35) using the BD FACSAria II Cell Sorter. Single-cell cDNA libraries were prepared using 10x Genomics technology, and the sequencing was performed on an Illumina NovaSeq 6000 platform, targeting 50000 reads per cell. Single cell RNA sequencing data was analyzed using RStudio software and Seurat packages. At d0 and d19 of EAM, bulk RNAseq was performed on live cardiac EGFP+CD45-CD34- fibroblasts. Results Following a quality control check, we included in the analysis 25058 cells. We identified 13 distinct main cell populations, including fibroblasts, lymphoid and myeloid immune cells, endothelial cells, smooth muscle cells, and cardiomyocytes (Fig. 1A). Differential expression analysis revealed that fibroblasts in acute myocarditis (d19) showed activation of genes related to immune processes such as cytokine-cytokine receptor interaction, IL-17, and TNF-alpha signaling pathways. Subset analysis on 4901 cardiac fibroblasts resulted in 5 distinct fibroblast subsets, FB1–FB5 (Fig. 1B). A major fibroblast subset found in the heart (FB1) expanded during the course of EAM (Fig. 1C). Of note, cells expressing the myofibroblastic signature (FB4) were identified at d19 only (Fig. 1C). Fibroblast subset FB1 activated expression of proinflammatory genes, such as Ly6a, Ly6c1, Cxcl14, Ccl2, and Ccl7, particularly during the inflammatory phase of EAM (d19, Fig. 1D). Surprisingly, the expression of profibrotic genes, including Col1a1, Col3a1, and Postn in FB1, was downregulated during the progression of EAM (Fig. 1E). Bulk RNA sequencing analysis confirmed that cardiac fibroblasts in acute myocarditis (d19) activated immune processes (chemokine production), but also showed dysregulated expression of genes involved in cytoskeletal re-organization and extracellular matrix turnover. Conclusions Our data indicated that acute inflammation in the heart activated a pro-inflammatory transcriptional response in cardiac fibroblasts and induced a fibroblast-to-myofibroblast transition. Understanding changes in transcriptomic signatures on a single cell level can help to develop new therapies in inflammatory heart diseases.

Abstract 17132: IRE1α Protects Against Cardiac Fibrosis via Regulating Selective Transcript Degradation

Background: Upon cardiac injury, cardiac fibroblasts become activated and differentiate intomyofibroblasts to maintain cardiac structure and function through secretion of stabilizingextracellular matrix (ECM) components. However, if this persists, it leads to stiffening of themyocardium and heart failure. The unfolded protein response (UPR) responds to an increaseddemand for folding of nascent secreted ECM proteins trafficked through the endoplasmicreticulum such as collagen. IRE1α, the most evolutionarily conserved arm of the UPR, actsadaptively both by enhancing protein folding of secreted proteins and reducing prolongedincreases in protein synthesis as occurs during pathologic cardiac fibrosis. Hypothesis: IRE1α protects against cardiac fibrosis via inhibiting pathologic sustained fibroblastactivation. Methods: Primary cardiac fibroblasts (CFBs) were cultured with transforming growth factor beta(TGF-β) to promote activation, in vitro . Small molecule targeting approaches were utilized tospecifically inhibit or activate IRE1α. IRE1α-floxed mice were injected with AAV9 to knockoutIRE1α specifically in myofibroblasts (IRE1mfbKO) and were subjected to transverse aorticconstriction (TAC) as a model of heart failure to stimulate fibroblast activation, in vivo . Results: While small molecule activation of IRE1α significantly blunted CFB activation inresponse to TGF-β, specific inhibition of IRE1α’s endonuclease domain enhanced CFBresponsiveness to TGF-β. Importantly, this effect occurred independent of IRE1α’s canonicalmechanism of activating the transcription factor, XBP1, implicating regulated IRE1α-dependentdecay (RIDD) as a novel anti-fibrotic protective mechanism. Transcriptomics of CFBs identifieda novel “RIDD-ome” of pro-fibrotic mRNA targets of IRE1α-mediated degradation. Finally, inresponse to TAC, IRE1mfbKO mice exhibited an exacerbated decrease in cardiac function andmarked increase interstitial fibrosis. Conclusions: IRE1α protects against pathologic differentiation of CFBs in response to TGF-β or TACvia regulating the degradation of a novel subset of pro-fibrotic transcripts. Thus, small moleculetargeting of IRE1α represents a novel approach to mitigating cardiac fibrosis.

Abstract 15535: SGK1 is a Key Mediator of Pathological Cardiac Fibrosis

Background: Cardiac fibroblasts initially respond to injury adaptively, proliferating and differentiating into myofibroblasts to maintain cardiac structure and function. However, prolonged cardiac stress leads to pathological cardiac fibrosis, ultimately resulting in heart failure (HF). Thus, there is a major interest in developing therapeutic strategies to constrain fibroblast differentiation and sustained activation. We recently identified the cytosolic protein kinase, serum/glucocorticoid regulated kinase 1 (SGK1), as being largely involved with pathological remodeling in the cardiac myocyte. While there have been reports that SGK1 may also play a role in fibrosis, the mechanistic role for SGK1 in cardiac fibroblasts in response to injury remains unknown. Hypothesis: SGK1 promotes pathological cardiac fibrosis and exacerbates progression to HF. Methods: Transforming growth factor beta, (TGFβ) was used in primary cardiac fibroblasts (CFBs), in vitro , and transverse aortic constriction (TAC) was used as a HF model in mice, in vivo . CFBs were transfected with siRNA to knockdown SGK1, in vitro , and SGK1 floxed mice were injected with a novel AAV9 utilizing a myofibroblast-specific promoter to knockout SGK1 specifically in myofibroblasts (SGK1 mfbKO), in vivo . Results: SGK1 signaling was elevated in cardiac explants obtained from ischemic human HF patients which led us to further evaluate SGK1 activity in CFBs. SGK1 knockdown in CFBs attenuated TGFβ-mediated differentiation and fibrotic signaling. SGK1 mfbKO mice subjected to TAC showed preserved systolic and diastolic cardiac function, measured by echocardiography. At the biochemical level, pathological remodeling and gene markers of fibrosis were attenuated in SGK1 mfbKO mice as determined by qRT-PCR of cardiac extracts. Finally, treatment of CFBs with a small molecule SGK1 inhibitor attenuated differentiation and fibrotic signaling in response to TGFβ treatment. Conclusion: SGK1 knockdown is protective against cardiac fibrosis, attenuating differentiation and fibrotic signaling in CFBs. Further, SGK1 mfbKO protected against pathological cardiac remodeling and functional decline in a mouse model of HF, highlighting the specific pathophysiological contributions of CFBs.

NKRF in Cardiac Fibroblasts Protects against Cardiac Remodeling Post-Myocardial Infarction via Human Antigen R.

Myocardial infarction (MI) remains the leading cause of death worldwide. Cardiac fibroblasts (CFs) are abundant in the heart and are responsible for cardiac repair post-MI. NF-κB-repressing factor (NKRF) plays a significant role in the transcriptional inhibition of various specific genes. However, the NKRF action mechanism in CFs remains unclear in cardiac repair post-MI. This study investigates the NKRF mechanism in cardiac remodeling and dysfunction post-MI by establishing a CF-specific NKRF-knockout (NKRF-CKO) mouse model. NKRF expression is downregulated in CFs in response to pathological cardiac remodeling in vivo and TNF-α in vitro. NKRF-CKO mice demonstrate worse cardiac function and survival and increased infarct size, heart weight, and MMP2 and MMP9 expression post-MI compared with littermates. NKRF inhibits CF migration and invasion in vitro by downregulating MMP2 and MMP9 expression. Mechanistically, NKRF inhibits human antigen R (HuR) transcription by binding to the classical negative regulatory element within the HuR promoter via an NF-κB-dependent mechanism. This decreases HuR-targeted Mmp2 and Mmp9 mRNA stability. This study suggests that NKRF is a therapeutic target for pathological cardiac remodeling.

Serelaxin inhibits LPS-induced inflammatory response in cardiac fibroblasts by activating PPAR-γ and suppressing NF-κB signaling pathway.

Serelaxin has an inhibitory effect on fibrosis. However, whether the anti-fibrotic effects of serelaxin are achieved by inhibiting the inflammatory response has not been clarified. This study aimed to investigate the role of serelaxin in lipopolysaccharide (LPS)-induced inflammation in cardiac fibroblasts (CFs) and elucidate the underlying mechanisms.CFs were isolated from adult rat hearts. The effect of serelaxin on the inhibition of the inflammatory response after LPS induction was examined. Cell viability was measured by MMT assay. Cell proliferation was determined using the cell-counting kit-8. The levels of inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-10 were measured using an enzyme-linked immunosorbent assay (ELISA). The mRNA levels of α-SMA, collagen I/III, MMP-2, MMP-9, IL-1β, IL-6,TNF-α, IL-10, IκBα, p-IκBα, p65 subunit of nuclear factor-kappa B (NF-κB) and peroxisome proliferator-activated receptor-γ (PPAR-γ) were assessed by real-time quantitative PCR (RT-qPCR). The protein levels of α-SMA, collagen I/III, MMP-2, MMP-9, IκBα, p-IκBα, p65, p-p65 and PPAR-γ were examined by western blotting.Serelaxin inhibited LPS-induced IL-1β, IL-6, TNF-α, α-SMA, and collagen I/III, and elevated the expression of IL-10, MMP-2 and MMP-9. Moreover, LPS-induced activation of NF-κB pathway was suppressed by serelaxin treatment. Further studies showed that serelaxin did not significantly increase the expression of PPAR-γ mRNA and protein, but activated PPAR-γ activity, and the PPAR-γ inhibitor GW9662 reversed the inhibitory effect of serelaxin on IL-1β, IL-6, and TNF-α production. These results suggest that serelaxin alleviates cardiac fibrosis by stimulating PPAR-γ via a ligand-independent mechanism which subsequently abolish the expression of NF-κB signalling pathway.

Abstract P1173: Deciphering CITED4-dependent Signaling In The Heart Through Nascent Proteomics And Secretomics

Objective: Neuregulin-1 (Nrg1) promotes cardiomyocyte hypertrophy, survival, and cell cycle activity through ErbB signaling. It is investigated in clinical trials as a cardioprotective agent; however, its therapeutic use might be limited due to its pro-neoplastic potential for non-cardiomyocytes that makes targeted approaches necessary. High-throughput screening of hypertrophic agonists found that Nrg1 induces CITED4 (C4) expression. C4 is also upregulated in both physiological and pathological cardiac growth and necessary to prevent adverse remodeling in vivo. However, how C4 regulates Nrg1-signaling in the heart is yet unknown. Methods: We combined pulsed-SILAC labeling, click-chemistry and mass spectrometry that allows to capture immediate changes in the proteome and secretome after Nrg1 (12h and 24h) stimulation in siRNA-mediated C4-knockdown (C4KD) and control (ctr) NRVM and complemented this with established cell- and molecular biology techniques to validate and investigate cellular function and molecular signaling. Results: We confirmed dose-dependent C4 mRNA upregulation in response to Nrg1 stimulation in NRVM. Nrg1 downstream signaling through AKT was hindered in C4KD. Computational analysis comparing the nascent proteome after Nrg1 stimulation in C4KD and ctr NRVM revealed that C4 significantly regulates proteins responsible for translation, RNA processing and energy derivation. Consistent with previously observed development of cardiac fibrosis in C4 knockout mice in vivo , we found significant upregulation of TGFb2 in C4KD NRVM. We next confirmed the upregulation of TGFb2 and its downstream effectors in C4KD and ctr, while adenoviral overexpression of C4 led to reduced TGFb2 expression. Additionally, we found TGFb2 secretion increased from C4KD NRVM with Nrg1 stimulation in the nascent secretome. Conditioned media from C4KD NRVM led to an increased pro-fibrotic response in cardiac fibroblasts. In conclusion, nascent proteomics and secretomics help to elucidate C4-dependent Nrg1-signaling, which may be an important contribution toward our goal to identify targetable cardioprotective pathways in the heart.

Retinoid X receptor agonists alleviate fibroblast activation and post-infarction cardiac remodeling via inhibition of TGF-β1/Smad pathway

Retinoid X receptor (RXR), particularly RXRα, has been implicated in cardiovascular diseases. However, the functional role of RXR activation in myocardial infarction (MI) remains unclear. This study aimed to determine the effects of RXR agonists on MI and to dissect the underlying mechanisms. Sprague-Dawley (SD) rats were subjected to MI and then treated (once daily for 4 weeks) with either RXR agonist bexarotene (10 or 30 mg/kg body weight) or vehicle. Heart function was determined using echocardiography and cardiac hemodynamic measurements. Four weeks post MI, myocardial tissues were collected to evaluate cardiac remodeling. Primary cardiac fibroblasts (CFs) were treated with or without RXR ligand 9-cis-RA followed by stimulation with TGF-β1. Immunoblot, immunofluorescence, and co-immunoprecipitation were performed to elucidate the regulatory role of RXR agonists in TGF-β1/Smad signaling. In vivo treatment with Bexarotene moderately affects systemic inflammation and apoptosis and ameliorated left ventricular dysfunction after MI in rat model. In contrast, bexarotene significantly inhibited post-MI myocardial fibrosis. Immunoblot analysis of heart tissue homogenates from MI rats revealed that bexarotene regulated the activation of the TGF-β1/Smad signaling pathway. In vitro, 9-cis-RA inhibited the TGF-β1-induced proliferation and collagen production of CFs. Importantly, upon activation by 9-cis-RA, RXRα interacted with p-Smad2 in cytoplasm, inhibiting the TGF-β1-induced nuclear translocation of p-Smad2, thereby negatively regulating TGF-β1/Smad signaling and attenuating the fibrotic response of CFs. These findings suggest that RXR agonists ameliorate post-infarction myocardial fibrosis, maladaptive remodeling, and heart dysfunction via attenuation of fibrotic response in CFs through inhibition of the TGF-β1/Smad pathway activation.

Exercise-induced myocardial hypertrophy preconditioning promotes fibroblast senescence and improves myocardial fibrosis through Nrf2 signaling pathway

ABSTRACT This study aims to investigate how exercise-induced myocardial hypertrophy preconditioning affects cardiac fibroblasts in the context of myocardial fibrosis, a chronic disease that can cause cardiac arrhythmia and heart failure. Heart failure was induced in male C57BL/6 mice via Transverse aortic constriction, and some mice were given swimming exercise before surgery to test the effects of exercise-induced myocardial hypertrophy preconditioning on myocardial fibrosis. Myocardial tissue was evaluated for fibrosis, senescent cells, and apoptotic cells. Myocardial fibroblasts from rats were cultured and treated with norepinephrine to induce fibrosis which were then treated with si-Nrf2 and analyzed for markers of fibrosis, senescence, apoptosis, and cell proliferation. Exercise-induced myocardial hypertrophy preconditioning reduced myocardial fibrosis in mice, as shown by decreased mRNA expression levels of fibrosis-related indicators and increased cell senescence. In vitro data indicated that norepinephrine (NE) treatment increased fibrosis-related markers and reduced apoptotic and senescent cells, and this effect was reversed by pre-conditioning in PRE+NE group. Preconditioning activated Nrf2 and downstream signaling genes, promoting premature senescence in cardiac fibroblasts and tissues isolated from preconditioned mice. Moreover, Nrf2 knockdown reversed proapoptotic effects, restored cell proliferation, reduced senescence-related protein expression, and increased oxidative stress markers and fibrosis-related genes, indicating Nrf2‘s crucial role in regulating oxidative stress response of cardiac fibroblasts. Exercise-induced myocardial hypertrophy preconditioning improves myocardial fibrosis which is Nrf2-dependent, indicating the protective effect of hypertrophy preconditioning. These findings may contribute to the development of therapeutic interventions to prevent or treat myocardial fibrosis.

Dectin-1 deficiency alleviates diabetic cardiomyopathy by attenuating macrophage-mediated inflammatory response

Cardiovascular diseases are the primary cause of mortality in patients with diabetes and obesity. Hyperglycemia and hyperlipidemia in diabetes alters cardiac function, which is associated with broader cellular processes such as aberrant inflammatory signaling. Recent studies have shown that a pattern recognition receptor called Dectin-1, expressed on macrophages, mediates pro-inflammatory responses in innate immunity. In the present study, we examined the role of Dectin-1 in the pathogenesis of diabetic cardiomyopathy. We observed increased Dectin-1 expression in heart tissues of diabetic mice and localized the source to macrophages. We then investigated the cardiac function in Dectin-1-deficient mice with STZ-induced type 1 diabetes and high-fat-diet-induced type 2 diabetes. Our results show that Dectin-1 deficient mice are protected against diabetes-induced cardiac dysfunction, cardiomyocyte hypertrophy, tissue fibrosis, and inflammation. Mechanistically, our studies show that Dectin-1 is important for cell activation and induction of inflammatory cytokines in high-concentration glucose and palmitate acid (HG + PA)-challenged macrophages. Deficiency of Dectin-1 generate fewer paracrine inflammatory factors capable of causing cardiomyocyte hypertrophy and fibrotic responses in cardiac fibroblasts. In conclusion, this study provides evidence that Dectin-1 mediates diabetes-induced cardiomyopathy through regulating inflammation. Dectin-1 may be a potential target to combat diabetic cardiomyopathy.

Lipoxin A4 prevents high glucose-induced inflammatory response in cardiac fibroblast through FOXO1 inhibition

Cardiac cells respond to various pathophysiological stimuli, synthesizing inflammatory molecules that allow tissue repair and proper functioning of the heart; however, perpetuation of the inflammatory response can lead to cardiac fibrosis and heart dysfunction. High concentration of glucose (HG) induces an inflammatory and fibrotic response in the heart. Cardiac fibroblasts (CFs) are resident cells of the heart that respond to deleterious stimuli, increasing the synthesis and secretion of both fibrotic and proinflammatory molecules. The molecular mechanisms that regulate inflammation in CFs are unknown, thus, it is important to find new targets that allow improving treatments for HG-induced cardiac dysfunction. NFκB is the master regulator of inflammation, while FoxO1 is a new participant in the inflammatory response, including inflammation induced by HG; however, its role in the inflammatory response of CFs is unknown. The inflammation resolution is essential for an effective tissue repair and recovery of the organ function. Lipoxin A4 (LXA4) is an anti-inflammatory agent with cytoprotective effects, while its cardioprotective effects have not been fully studied. Thus, in this study, we analyze the role of p65/NFκB, and FoxO1 in CFs inflammation induced by HG, evaluating the anti-inflammatory properties of LXA4. Our results demonstrated that HG induces the inflammatory response in CFs, using an in vitro and ex vivo model, while FoxO1 inhibition and silencing prevented HG effects. Additionally, LXA4 inhibited the activation of FoxO1 and p65/NFκB, and inflammation of CFs induced by HG. Therefore, our results suggest that FoxO1 and LXA4 could be novel drug targets for the treatment of HG-induced inflammatory and fibrotic disorders in the heart.

Insulin-like growth factor-1 production by neonatal mouse cardiac fibroblasts in response to growth hormone is defective and dispensable for hypertrophy of neonatal mouse cardiomyocytes

Insulin-like growth factor-1 production by neonatal mouse cardiac fibroblasts in response to growth hormone is defective and dispensable for hypertrophy of neonatal mouse cardiomyocytes

HuR-dependent expression of Wisp1 is necessary for TGFβ-induced cardiac myofibroblast activity

Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGFβ-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGFβ-induced myofibroblast activation upon HuR inhibition. We identified twenty-four mRNA transcripts that were enriched for HuR binding following TGFβ treatment via photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). Eleven of these HuR-bound mRNAs also showed significant co-expression correlation with HuR, αSMA, and periostin in primary fibroblasts isolated from the ischemic-zone of infarcted mouse hearts. Of these, WNT1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 partially rescued myofibroblast-induced collagen gel contraction following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGFβ. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGFβ, in part through post-transcriptional regulation of Wisp1.

Unveiling the Pathologic Response of Cardiac Fibroblasts During Aspergillus fumigatus Pulmonary Infections

Various adverse cardiovascular events have been associated with lung damage as a result of pathological infections followed by chronic inflammation. In this context, several recent studies report severe cardiovascular complications after pulmonary infection by SARS‐CoV‐2. Therefore, determining the physiological link between abnormal pulmonary and cardiac function is an emerging clinical need in the cardiovascular field. The main limitation of these studies has been the lack of specific genetic tools and mouse models to study the cellular and molecular mechanisms that regulate the lung‐heart interactions. To induce lung damage we utilized a common airborne environmental fungus, Aspergillus fumigatus (AF), which is an opportunistic pathogen that establishes pathological infection in the lung if the immune system is compromised. However, in immunocompetent animals, transient stress to the lung is still detectable before clearance via immune cells. Our lab has discovered that pulmonary fibroblasts (PFs), typically associated with the maintenance and production of the extracellular matrix, activate in response to inhaled AF spores and modulate the immune response. Moreover, we have also observed activation of cardiac fibroblasts (CFs) in response to the pulmonary AF infection. Therefore, we hypothesized that pulmonary infection leads to cardiac stress, resulting in the activation and pathological response of CFs, and subsequently result in myocardial remodeling, fibrosis, and disease. To test our hypothesis we used our recently developed and described in vivo cre‐loxP lineage tracing mouse lines to specifically tag and manipulate all activated fibroblast during infection, clearing and reinfection. Our data show that CFs can activate after an acute inhalation of AF spores which are quickly cleared in immunocompetent mice with no pathological lung damage. Moreover, subsequent asymptomatic chronic AF infections result in the accumulation of PF and CF pathologic activation. These results reveal a cellular novel link between pulmonary infection and its impact on cardiovascular events.

Activated myofibroblasts promote cardiac hypertrophy and systolic dysfunction independently of cardiac fibrosis in experimental autoimmune myocarditis

Abstract Background/Introduction Heart-specific inflammation – myocarditis is a common cause dilated cardiomyopathy which is characterized by pathological tissue remodeling, ventricular stiffening, cardiomyopathy and heart failure. In experimental autoimmune myocarditis (EAM) susceptible mice immunized with alpha myosin heavy chain (αMyHC) and complete Freund's adjuvant (CFA) develop acute myocarditis driven by autoreactive CD4+ T cells that is followed by progressive fibrosis, cardiomyopathy and systolic dysfunction. Purpose The aim of the study was to investigate the role of cardiac fibroblasts and myofibroblasts in myocarditis and post-inflammatory dilated cardiomyopathy in mouse model of EAM. Methods EAM was induced in BALB/c mice by immunization with αMyHC/CFA. We used reporter mice expressing EGFP under collagen type I promoter (Coll-EGFP) and RFP under a control of α-smooth muscle actin (αSMA) promoter (αSMA-RFP) and transgenic αSMA-TK mice with ganciclovir-inducible ablation of proliferating myofibroblasts. Cardiac cells were quantified using flow cytometry. Cardiac fibroblasts (CD45-CD31-EGFP+) were sorted from healthy and myocarditis-positive (day 21) mice using BD FACSAria™ II Cell Sorter and analyzed for the whole genome transcriptomics by RNA sequencing. Echocardiography was performed on Vevo 2100 Imaging System. Cardiac fibrosis was assessed by Trichrome Massons's staining and hydroxyproline assay, whereas cardiac hypertrophy by analysing cross-sectional cardiomyocyte area. Profibrotic gene expression was assessed by qRT-PCR. Results The total number of cardiac fibroblasts (CD45-CD31-EGFP+) and the subset of myofibroblasts (CD45-CD31-EGFP+RFP+) remained unchanged at inflammatory (d21) and fibrotic stages (d40). Analysis of differentially expressed genes (min. 2x fold change, p value <0.05) pointed out activation of immune processes (mainly chemokine production), response to stress, cytoskeletal and extracellular matrix re-organization in cardiac fibroblasts in response to myocarditis. αSMA-TK mice treated with ganciclovir (from day 21) showed comparable percent of fibrotic area, but significantly reduced heart weight, decreased cardiomyocyte hypertrophy and improved ejection fraction and cardiac output at day 40 comparing to PBS-treated mice. Ganciclovir-treated mice showed also attenuated cardiac Acta2 and Srf but markedly enhanced Mmp2 expression. Conclusions In EAM model cardiac fibroblasts actively participate in proinflammatory and profibrotic responses, while activated myofibroblasts contribute to dilated cardiomyopathy development independently of cardiac fibrosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre (Poland)

A Catecholaldehyde Metabolite of Norepinephrine Induces Myofibroblast Activation and Toxicity via the Receptor for Advanced Glycation Endproducts: Mitigating Role of l-Carnosine.

Monoamine oxidase (MAO) is rapidly gaining appreciation for its pathophysiologic role in cardiac injury and failure. Oxidative deamination of norepinephrine by MAO generates H2O2 and the catecholaldehyde 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), the latter of which is a highly potent and reactive electrophile that has been linked to cardiotoxicity. However, many questions remain as to whether catecholaldehydes regulate basic physiological processes in the myocardium and the pathways involved. Here, we examined the role of MAO-derived oxidative metabolites in mediating the activation of cardiac fibroblasts in response to norepinephrine. In neonatal murine cardiac fibroblasts, norepinephrine increased reactive oxygen species (ROS), accumulation of catechol-modified protein adducts, expression and secretion of collagens I/III, and other markers of profibrotic activation including STAT3 phosphorylation. These effects were attenuated with MAO inhibitors, the aldehyde-scavenging dipeptide l-carnosine, and FPS-ZM1, an antagonist for the receptor for advanced glycation endproducts (RAGE). Interestingly, treatment of cardiac fibroblasts with a low dose (1 μM) of DOPEGAL-modified albumin phenocopied many of the effects of norepinephrine and also induced an increase in RAGE expression. Higher doses (>10 μM) of DOPEGAL-modified albumin were determined to be toxic to cardiac fibroblasts in a RAGE-dependent manner, which was mitigated by l-carnosine. Collectively, these findings suggest that norepinephrine may influence extracellular matrix remodeling via an adrenergic-independent redox pathway in cardiac fibroblasts involving the MAO-mediated generation of ROS, catecholaldehydes, and RAGE. Furthermore, since elevations in the catecholaminergic tone and oxidative stress in heart disease are linked with cardiac fibrosis, this study illustrates novel drug targets that could potentially mitigate this serious disorder.

Effects of Sex and 17 β-Estradiol on Cardiac Fibroblast Morphology and Signaling Activities In Vitro.

Several studies have demonstrated estrogen’s cardioprotective abilities in decreasing the fibrotic response of cardiac fibroblasts (CFs). However, the majority of these studies are not sex-specific, and those at the cellular level utilize tissue culture plastic, a substrate with a much higher stiffness than physiological conditions. Understanding the intrinsic differences between male and female CFs under more physiologically “healthy” conditions will help to elucidate the divergences in their complex signaling networks. We aimed to do this by conducting a sex-disaggregated analysis of changes in cellular morphology and relative levels of profibrotic signaling proteins in CFs cultured on 8 kPa stiffness plates with and without 17 β-estradiol (E2). Cyclic immunofluorescent analysis indicated that there was a negligible change in cellular morphology due to sex and E2 treatment and that the differences between male and female CFs occur at a biochemical rather than structural level. Several proteins corresponding to profibrotic activity had various sex-specific responses with and without E2 treatment. Single-cell correlation analysis exhibited varied protein–protein interaction across experimental conditions. These findings demonstrate the need for further research into the dimorphisms of male and female CFs to develop better tailored sex-informed prevention and treatment interventions of cardiac fibrosis.

WNT/β-Catenin Signaling Promotes TGF-β-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production.

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-β and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-β-activated human cardiac fibroblasts. We found that WNT3a and TGF-β induced a β-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-β triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the β-catenin pathway with the GSK3β inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-β, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-β-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-β and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-β-induced production of collagen I. In conclusion, WNT/β-catenin signaling promotes TGF-β-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.