Changes In Cardiac Fibroblasts Research Articles

Multi-level transcriptomic analysis of LMNA -related dilated cardiomyopathy identifies disease-driving processes.

LMNA- related dilated cardiomyopathy ( LMNA -DCM) is one of the most severe forms of DCM. The incomplete understanding of the molecular disease mechanisms results in lacking treatment options, leading to high mortality amongst patients. Here, using an inducible, cardiomyocyte-specific lamin A/C depletion mouse model, we conducted a comprehensive transcriptomic study, combining both bulk and single nucleus RNA sequencing, and spanning LMNA -DCM disease progression, to identify potential disease drivers. Our refined analysis pipeline identified 496 genes already misregulated early in disease. The expression of these genes was largely driven by disease specific cardiomyocyte sub-populations and involved biological processes mediating cellular response to DNA damage, cytosolic pattern recognition, and innate immunity. Indeed, DNA damage in LMNA -DCM hearts was significantly increased early in disease and correlated with reduced cardiomyocyte lamin A levels. Activation of cytosolic pattern recognition in cardiomyocytes was independent of cGAS, which is rarely expressed in cardiomyocytes, but likely occurred downstream of other pattern recognition sensors such as IFI16. Altered gene expression in cardiac fibroblasts and immune cell infiltration further contributed to tissue-wide changes in gene expression. Our transcriptomic analysis further predicted significant alterations in cell-cell communication between cardiomyocytes, fibroblasts, and immune cells, mediated through early changes in the extracellular matrix (ECM) in the LMNA -DCM hearts. Taken together, our work suggests a model in which nuclear damage in cardiomyocytes leads to activation of DNA damage responses, cytosolic pattern recognition pathway, and other signaling pathways that activate inflammation, immune cell recruitment, and transcriptional changes in cardiac fibroblasts, which collectively drive LMNA -DCM pathogenesis.

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.

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.

Phenotypic characterization of primary cardiac fibroblasts from patients with HFpEF.

Heart failure is a leading cause of hospitalizations and mortality worldwide. Heart failure with a preserved ejection fraction (HFpEF) represents a significant clinical challenge due to the lack of available treatment modalities for patients diagnosed with HFpEF. One symptom of HFpEF is impaired diastolic function that is associated with increases in left ventricular stiffness. Increases in myocardial fibrillar collagen content is one factor contributing to increases in myocardial stiffness. Cardiac fibroblasts are the primary cell type that produce fibrillar collagen in the heart. However, relatively little is known regarding phenotypic changes in cardiac fibroblasts in HFpEF myocardium. In the current study, cardiac fibroblasts were established from left ventricular epicardial biopsies obtained from patients undergoing cardiovascular interventions and divided into three categories: Referent control, hypertension without a heart failure designation (HTN (-) HFpEF), and hypertension with heart failure (HTN (+) HFpEF). Biopsies were evaluated for cardiac myocyte cross-sectional area (CSA) and collagen volume fraction. Primary fibroblast cultures were assessed for differences in proliferation and protein expression of collagen I, Membrane Type 1-Matrix Metalloproteinase (MT1-MMP), and α smooth muscle actin (αSMA). Biopsies from HTN (-) HFpEF and HTN (+) HFpEF exhibited increases in myocyte CSA over referent control although only HTN (+) HFpEF exhibited significant increases in fibrillar collagen content. No significant changes in proliferation or αSMA was detected in HTN (-) HFpEF or HTN (+) HFpEF cultures versus referent control. Significant increases in production of collagen I was detected in HF (-) HFpEF fibroblasts, whereas significant decreases in MT1-MMP levels were measured in HTN (+) HFpEF cells. We conclude that epicardial biopsies provide a viable source for primary fibroblast cultures and that phenotypic differences are demonstrated by HTN (-) HFpEF and HTN (+) HFpEF cells versus referent control.

The landscape of accessible chromatin in quiescent cardiac fibroblasts and cardiac fibroblasts activated after myocardial infarction

ABSTRACT After myocardial infarction, the massive death of cardiomyocytes leads to cardiac fibroblast proliferation and myofibroblast differentiation, which contributes to the extracellular matrix remodelling of the infarcted myocardium. We recently found that myofibroblasts further differentiate into matrifibrocytes, a newly identified cardiac fibroblast differentiation state. Cardiac fibroblasts of different states have distinct gene expression profiles closely related to their functions. However, the mechanism responsible for the gene expression changes during these activation and differentiation events is still not clear. In this study, the gene expression profiling and genome-wide accessible chromatin mapping of mouse cardiac fibroblasts isolated from the uninjured myocardium and the infarct at multiple time points corresponding to different differentiation states were performed by RNA sequencing (RNA-seq) and the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), respectively. ATAC-seq peaks were highly enriched in the promoter area and the distal area where the enhancers are located. A positive correlation was identified between the expression and promoter accessibility for many dynamically expressed genes, even though evidence showed that mechanisms independent of chromatin accessibility may also contribute to the gene expression changes in cardiac fibroblasts after MI. Moreover, motif enrichment analysis and gene regulatory network construction identified transcription factors that possibly contributed to the differential gene expression between cardiac fibroblasts of different states.

Abstract 14422: Molecular Pathogenesis of Cardiac Microthrombi in Fatal Covid-19: Insights from Clinico-histopathologic and Single Nuclei RNA Sequencing Analyses

Cardiac microthrombi are postulated to underlie cardiac injury in critical COVID-19. To determine pathogenic mechanism(s) of cardiac injury in fatal COVID-19, we conducted a single-center prospective cohort study of 69 consecutive COVID-19 decedents. Microthrombi was the most commonly detected acute cardiac histopathologic feature (n=48, 70%). We tested associations of cardiac microthrombi with biomarkers of inflammation, cardiac injury, and fibrinolysis and with in-hospital antiplatelet therapy, therapeutic anticoagulation, and corticosteroid treatment, while adjusting for multiple clinical factors, including COVID-19 therapies. Higher peak ESR and CRP during hospitalization were independently associated with higher odds of microthrombi (ESR, P non-linearity 0.015, P association =0.008; CRP per 20mg/L increase, OR 1.17, 95%CI 1.00-1.36). Using single nuclei RNA-sequence analysis, we discovered an enrichment of prothrombotic, anti-fibrinolytic, and extracellular matrix signaling amongst cardiac fibroblasts in microthrombi-positive COVID-19 hearts, compared with microthrombi-negative COVID-19 hearts and non-COVID-19 donor hearts. Our cumulative findings identify these specific transcriptomic changes in cardiac fibroblasts as salient features of COVID-19-associated cardiac microthrombi.

Inhibition of the mevalonate pathway improves myocardial fibrosis.

The mevalonate (MVA) pathway serves an important role in ventricular remodeling. Targeting the MVA pathway has protective effects against myocardial fibrosis. The present study aimed to investigate the mechanism behind these effects. Primary cultured cardiac fibroblasts from C57BL/6 mice were treated in vitro in 5 groups: i) negative control; ii) angiotensin II (Ang II) model (1x10-5 mol/l); iii) Ang II + rosuvastatin (ROS); iv) Ang II + alendronate (ALE); and v) Ang II + fasudil (FAS). Collagen and crystal violet staining were used to assess morphological changes in cardiac fibroblasts. Reverse transcription quantitative PCR and western blotting were used to analyze the expression of key signaling molecules involved in the MVA pathway. Collagen staining in the ALE, FAS, and ROS groups was weak compared with the Ang II group, while the rate of cell proliferation in the ROS, ALE, and FAS groups was slower compared with that in the Ang II group. In addition, the expression of key signaling molecules in the MVA pathway, including transforming growth factor-β1 (TGF-β1), heat shock protein 47 (HSP47), collagen type I α1 (COL1A1), vascular endothelial growth factor 2 (VEGF2) and fibroblast growth factor 2 (FGF2), was decreased in the FAS and ROS groups compared with the Ang II model. Compared with the Ang II group, 3-Hydroxy-3-Methylglutaryl-CoA reductase (HMGCR) gene expression was significantly lowered in the drug intervention groups, whereas farnesyl pyrophosphate synthase (FDPS) expression was downregulated in the ALE group, but elevated in the FAS and ROS groups. Compared with that in the Ang II group, ras homolog family member A (RhoA) expression was downregulated in the FAS and ROS groups, whilst mevalonate kinase expression was reduced in the ROS group. Protein expression of TGF-β1, COL1A1 and HSP47 were decreased following intervention with each of the three drugs compared with the Ang II group. Overall, rosuvastatin, aledronate and fasudil decreased the proliferation of myocardial fibroblasts and inhibited collagen synthesis. Rosuvastatin had the strongest protective effects against myocardial fibrosis compared with the other drugs tested, suggesting this to be a potential agent for the clinical treatment of cardiovascular disease.

CircUbe3a from M2 macrophage-derived small extracellular vesicles mediates myocardial fibrosis after acute myocardial infarction

Objective: This study aimed to explore the role of circular RNAs (circRNAs) in M2 macrophage (M2M)-derived small extracellular vesicles (SEVs) in myocardial fibrosis development.Methods: The regulatory role of M2M-derived extracellular vesicles (EVs) was evaluated in a mouse model of acute myocardial infarction. Immunofluorescence, quantitative real-time PCR (RT-qPCR), nanoparticle tracking analysis, Western blot analysis and electron microscopy were used to identify macrophages, large extracellular vesicles (LEVs) and SEVs. The circRNA expression profiles of M0 macrophages (M0Ms) and M2Ms were determined by microarray analysis. Bioinformatic analysis, cell coculture and cell proliferation assays were performed to investigate the expression, function, and regulatory mechanisms of circUbe3a in vitro. qPCR, RNA immunoprecipitation (RIP), dual-luciferase reporter assays, RNA fluorescence in situ hybridization (RNA-FISH), Western blot analysis and a series of rescue experiments were used to verify the correlation among circUbe3a, miR-138-5p and RhoC.Results: CircUbe3a from M2M-derived SEVs triggered functional changes in cardiac fibroblasts (CFs). CircUbe3a was synthesized and loaded into SEVs during increased M2M infiltration after myocardial infarction. The fusion of the released SEVs with the plasma membrane likely caused the release of circUbe3a into the cytosol of CFs. Silencing or overexpressing circUbe3a altered CF proliferation, migration, and phenotypic transformation in vitro. We confirmed that circUbe3a plays a crucial role in enhancing functional changes in CFs by sponging miR-138-5p and then translationally repressing RhoC in vitro. In vivo, the addition of M2M-derived SEVs or overexpression of circUbe3a significantly exacerbated myocardial fibrosis after acute myocardial infarction, and these effects were partially abolished by circUbe3a-specific shRNA.Conclusions: Our findings suggest that M2M-derived circUbe3a-containing SEVs promote the proliferation, migration, and phenotypic transformation of CFs by directly targeting the miR-138-5p/RhoC axis, which may also exacerbate myocardial fibrosis after acute myocardial infarction.

Activated Cardiac Fibroblasts Control Contraction of Human Fibrotic Cardiac Microtissues by a β-Adrenoreceptor-Dependent Mechanism.

Cardiac fibrosis represents a serious clinical problem. Development of novel treatment strategies is currently restricted by the lack of the relevant experimental models in a human genetic context. In this study, we fabricated self-aggregating, scaffold-free, 3D cardiac microtissues using human inducible pluripotent stem cell (iPSC)-derived cardiomyocytes and human cardiac fibroblasts. Fibrotic condition was obtained by treatment of cardiac microtissues with profibrotic cytokine transforming growth factor β1 (TGF-β1), preactivation of foetal cardiac fibroblasts with TGF-β1, or by the use of cardiac fibroblasts obtained from heart failure patients. In our model, TGF-β1 effectively induced profibrotic changes in cardiac fibroblasts and in cardiac microtissues. Fibrotic phenotype of cardiac microtissues was inhibited by treatment with TGF-β-receptor type 1 inhibitor SD208 in a dose-dependent manner. We observed that fibrotic cardiac microtissues substantially increased the spontaneous beating rate by shortening the relaxation phase and showed a lower contraction amplitude. Instead, no changes in action potential profile were detected. Furthermore, we demonstrated that contraction of human cardiac microtissues could be modulated by direct electrical stimulation or treatment with the β-adrenergic receptor agonist isoproterenol. However, in the absence of exogenous agonists, the β-adrenoreceptor blocker nadolol decreased beating rate of fibrotic cardiac microtissues by prolonging relaxation time. Thus, our data suggest that in fibrosis, activated cardiac fibroblasts could promote cardiac contraction rate by a direct stimulation of β-adrenoreceptor signalling. In conclusion, a model of fibrotic cardiac microtissues can be used as a high-throughput model for drug testing and to study cellular and molecular mechanisms of cardiac fibrosis.

Doxorubicin induces trans-differentiation and MMP1 expression in cardiac fibroblasts via cell death-independent pathways.

Although doxorubicin (DOX)-induced cardiomyopathy causes lethal heart failure (HF), no early detection or effective treatment methods are available. The principal mechanisms of cardiotoxicity are considered to involve oxidative stress and apoptosis of cardiomyocytes. However, the effect of DOX on cardiac fibroblasts at non-lethal concentrations remains unknown. The aim of this study was to investigate the direct effect of doxorubicin on the activation of cardiac fibroblasts independent of cell death pathways. We first found that DOX induced α-SMA expression (marker of trans-differentiation) at a low concentration range, which did not inhibit cell viability. DOX also increased MMP1, IL-6, TGF-β and collagen expression in human cardiac fibroblasts (HCFs). In addition, DOX promoted Akt and Smad phosphorylation. A Smad inhibitor prevented DOX-induced α-SMA and IL-6 protein expression. An PI3K inhibitor also prevented MMP1 mRNA expression in HCFs. These findings suggest that DOX directly induces fibrotic changes in HCFs via cell death-independent pathways. Furthermore, we confirmed that these responses are organ- and species-specific for HCFs based on experiments using different types of human and murine fibroblast cell lines. These results suggest potentially new mechanisms of DOX-induced cardiotoxicity from the viewpoint of fibrotic changes in cardiac fibroblasts.

Acute Hyperthermia Inhibits TGF-\u03b21-induced Cardiac Fibroblast Activation via Suppression of Akt Signaling

Transforming growth factor-β1 (TGF-β1) induces phenotypic changes in fibroblasts to become myofibroblasts with increased production of extracellular matrix (ECM) components and cytokines. It is also known that excessive activation of myofibroblasts accelerates cardiac fibrosis, remodeling, and thus cardiac dysfunction. However, no effective therapy has been established to prevent this process although recent clinical studies have demonstrated the effectiveness of hyperthermia in cardiac dysfunction. The aim of this study was to examine the molecular mechanism of hyperthermia on TGF-β1-mediated phenotypic changes in cardiac fibroblasts. TGF-β1 increased the expression of IL-6, α-smooth muscle actin (α-SMA), and collagen in human cardiac fibroblasts (HCFs). Hyperthermia (42 °C) significantly prevented these changes, i.e., increases in IL-6, α-SMA, and collagen, as induced by TGF-β1 in a time-dependent manner. Immunoblotting showed that hyperthermia decreased Akt/S6K signaling, but did not affect Smad2 and Smad3 signaling. Pharmacological inhibition of Akt signaling mimicked these effects of hyperthermia. Furthermore, hyperthermia treatment prevented cardiac fibrosis in Ang II infusion mice model. Putting together, our findings suggest that hyperthermia directly inhibits TGF-β-mediated activation of HCFs via suppressing Akt/S6K signaling.

TRPM7 channels mediate the functional changes in cardiac fibroblasts induced by angiotensin II.

Transient receptor potential melastatin7 (TRPM7), a bifunctional channel protein owning both cation permeability and kinase activity, plays an important role in the pathophysiological process of many cell types, such as vascular smooth muscle cells, human glioma cells and mouse cortical astrocytes. However, whether TRPM7 channels play a key role in the functional change of cardiac fibroblasts(CFs) induced by angiotensinII (AngII) remains unknown. Using Cell Counting Kit-8 (CCK-8) assay, immunofluorescence assay, western blot analysis, RT-qPCR, RNA interference(RNAi) and whole-cell patch-clamp techniques, the present study aimed to explore the role of TRPM7 channels in the proliferation, differentiation and collagen synthesis of CFs induced by AngII. Our data showed that AngII time-dependently increased TRPM7 expression and TRPM7 currents in the CFs. Downregulation of TRPM7 attenuated the TRPM7 current density, and inhibited the proliferation, differentiation and collagen synthesis of CFs induced by AngII. Our results identified the TRPM7 channel as a pivotal member associated with the functional change of CFs induced by AngII, and suggest that the TRPM7 channel may represent a promising therapeutic strategy for the treatment of fibrosis-related cardiac diseases.

Levosimendan inhibits interleukin-1β-induced apoptosis through activation of Akt and inhibition of inducible nitric oxide synthase in rat cardiac fibroblasts

Levosimendan, a calcium sensitizer, known as an inotropic agent has a cytoprotective effect against apoptosis in cardiomyocytes. However, the cytoprotective effect of levosimendan on cardiac fibroblasts has not been clarified. The aim of this study was to examine whether levosimendan modulates interleukin (IL)-1β-induced apoptosis in adult rat cardiac fibroblast. Cardiac fibroblasts were isolated from adult male Wistar rats. Apoptosis of cardiac fibroblasts was evaluated by 4′,6-diamidino-2-phenylindole staining and TdT-mediated d-UTP nick end labeling (TUNEL)-based staining. Expression of inducible nitric oxide (NO) synthase (iNOS) and phosphorylation of Akt (Ser473) were determined by Western blotting. Assessment of NO production in the culture medium was performed by using 4,5-diaminofluorescein as a fluorescent probe. Immunofluorescence staining was performed to examine cytochrome-c translocation. Levosimendan (3–100μM) concentration-dependently inhibited IL-1β (4ng/ml, 24h)-induced apoptotic changes in cardiac fibroblasts, such as cellular shrinkage, nuclear condensation and the increase of TUNEL-positive cells. Levosimendan inhibited IL-1β-induced iNOS expression and subsequent NO production. 1400W, an iNOS inhibitor, suppressed the IL-1β-induced apoptosis. Levosimendan alone-treatment concentration-dependently increased phosphorylation of Akt (Ser473). LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, reversed the protective effect of levosimendan on IL-1β-induced apoptosis in TUNEL assay. In addition, levosimendan and 1400W inhibited the IL-1β-induced cytosolic translocation of cytochrome-c. LY294002 reversed the suppressive effects of levosimendan. The present study for the first time demonstrated that levosimendan inhibits IL-1β-induced apoptosis via the activation of PI3K/Akt pathway and the inhibition of iNOS expression in adult rat cardiac fibroblasts.

Cardioprotective effects of prior transient ACE inhibition ő role of cardiac fibroblast (1152.7)

Transient ACE inhibition induces persistent changes that protect against future L‐NAME‐induced cardiac fibrosis and inflammation. This study investigates whether these observations can be mechanistically linked to changes in cardiac fibroblasts.We measured cardiac levels of chemokines, macrophages, and proliferating cells following 7 days of L‐NAME (L) in SHR that were previously treated with placebo (C+L) or enalapril (E+L) for 2wks followed by 2wk washout. Cardiac fibroblasts were isolated and cultured to passage 1.L‐NAME induced microinfarcts and vascular injury in both groups. In C+L, but not E+L there was an increase in macrophage‐recruiting chemokines. By day 7 of L‐NAME there was a marked increase in cardiac macrophages and proliferating cells in C+L, but not E+L. In vitro, fibroblasts from C+L were hyperproliferative, demonstrated increased Collagen I gene expression, and increased MCP‐1 production.These findings demonstrate that L‐NAME produces phenotypic changes in fibroblasts that persist in vitro that suggest that fibroblasts may participate in macrophage recruitment in addition to collagen production. However, fibroblasts isolated from SHR previously treated with enalapril display a different phenotype that more closely resembles cells from control hearts. ACE inhibitor‐induced cardioprotection may result, in part, from a modification of the cardiac fibroblast population.Grant Funding Source: Supported by: Sarver Heart Foundation, Steven M Gootter Foundation

TRPM7 is involved in angiotensin II induced cardiac fibrosis development by mediating calcium and magnesium influx

Cardiac fibrosis is involved in a lot of cardiovascular pathological processes. Cardiac fibrosis can block conduction, cause hypoxia, strengthen myocardial stiffness, create electrical heterogeneity, and hamper systolic ejection, which is associated with the development of arrhythmia, heart failure and sudden cardiac death. Besides the initial stimulating factors, the cardiac fibroblasts (CFs) are the principal responsible cells in the fibrogenesis cascade of events. TRPM7, a member of the TRPM (Melastatin) subfamily, is a non-selective cation channel, which permeates both Ca2+ and Mg2+. Here we demonstrated TRPM7 expression in CFs, and 2-APB (TRPM7 inhibitor), inhibited Ang II-induced CTGF, α-SMA expression and CFs proliferation. Besides, knocking down TRPM7 by shRNA, we proved that TRPM7 mediated both calcium and magnesium changes in cardiac fibroblasts which contribute to fibrosis progress. This study suggested that TRPM7 should play a pivotal role in cardiac fibroblast functions associated to cardiac fibrosis development.

Levosimendan inhibits interleukin-1β-induced cell migration and MMP-9 secretion in rat cardiac fibroblasts

Cell migration and matrix metalloproteinases (MMPs) secretion in cardiac fibroblasts are important processes in the cardiac remodeling during the development of cardiac diseases and are regulated by proinflammatory cytokines. Although levosimendan, a novel inotropic agent, is expected to have some beneficial influences on preventing cardiac remodeling, its effects on proinflammatory cytokines-induced functional changes in cardiac fibroblasts have not been clarified. Therefore, we investigated the effects of levosimendan on interleukin (IL)-1β−induced MMP-9 secretion and migration in adult rat cardiac fibroblasts. Primary cardiac fibroblasts were isolated from adult male Wistar rats. MMP-9 secretion in culture medium and extracellular signal-regulated kinase (ERK) phosphorylation in cell lysate were measured by using Western blotting. Gelatin zymography was performed to measure activity of secreted MMP-9. MMP-9 mRNA expression in the cell was measured by using reverse transcription polymerase chain reaction. Boyden chamber assay was performed for detection of migration. Levosimendan (3–100μM) concentration-dependently inhibited IL-1β (4ng/ml)-induced MMP-9 secretion, activity and mRNA expression. Levosimendan inhibited IL-1β (4ng/ml)-induced ERK phosphorylation. Levosimendan (10 and 100μM) inhibited IL-1β-induced migration, and CTTHWGFTLC peptide (10μM), an MMP inhibitor, or PD98059 (50μM), an ERK inhibitor, also suppressed it. The present study for the first time demonstrated in adult rat cardiac fibroblasts that levosimendan inhibits IL-1β-induced migration at least partly through the inhibition of MMP-9 secretion via suppressing ERK phosphorylation.

Abstract 310: Role of Cardiac Fibroblast in Cardioprotective Effects of Prior Transient ACE Inhibition

Prior treatment with the ACE inhibitor enalapril followed by washout protects against nitric oxide synthase inhibitor (L-NAME) induced fibrosis, cellular proliferation, and cardiac dysfunction. The present study investigated i) whether in vivo L-NAME administration induces a change in cardiac fibroblast phenotype that persists in vitro, ii) whether prior ACE inhibition protects against L-NAME induced changes in cardiac fibroblasts. SHR were divided into 3 groups: Control, L-NAME (C+L: 7d), enalapril+L-NAME (E+L: 14d enalapril + 14d washout + 7d L-NAME). MAP was measured by radiotelemetry (n=5-9), injury assessed by histology (n=6-10), and heart weight to body weight (HW/BW) was determined after 0 or 7 days of L-NAME in C+L and E+L (n=6-10). In separate rats cardiac fibroblasts were isolated after 7 days of L-NAME (C+L, E+L) or placebo (Con) and cultured to passage 1 (n=10-12). Gene expression was measured by quantitative real-time PCR. L-NAME increased MAP in C+L (22±4.1%) and E+L (21±3.6%) rats. Prior enalapril induced a persistent 13% reduction in HW/BW. L-NAME increased heart mass in E+L (7%) but not C+L; however, HW/BW remained 8% lower than C+L at sacrifice. L-NAME induced infarct in 70% of C+L and 40% of E+L hearts. Cardiac fibroblasts demonstrated a significant increase in proliferation rate in C+L, but not E+L, relative to control (C+L: 1.75-fold vs. con; E+L 1.09-fold vs. con). Fibroblasts from C+L hearts tended to have increased Collagen I and III gene expression. Despite hypertension, cardiac injury, and increased HW/BW; fibroblasts isolated from E+L proliferated at the same rate as those from control. In contrast, those isolated from C+L were hyperproliferative with a tendency toward increased capacity for collagen production. It may be that the fibroblast phenotype from E+L hearts would protect against infarct expansion and account, in part, for the previously reported cardioprotection in these rats.

Regulation of RhoA Signaling by the cAMP-dependent Phosphorylation of RhoGDIα

RhoA plays a pivotal role in regulating cell shape and movement. Protein kinase A (PKA) inhibits RhoA signaling and thereby induces a characteristic morphological change, cell rounding. This has been considered to result from cAMP-induced phosphorylation of RhoA at Ser-188, which induces a stable RhoA-GTP-RhoGDIα complex and sequesters RhoA to the cytosol. However, few groups have shown RhoA phosphorylation in intact cells. Here we show that phosphorylation of RhoGDIα but not RhoA plays an essential role in the PKA-induced inhibition of RhoA signaling and in the morphological changes using cardiac fibroblasts. The knockdown of RhoGDIα by siRNA blocks cAMP-induced cell rounding, which is recovered by RhoGDIα-WT expression but not when a RhoGDIα-S174A mutant is expressed. PKA phosphorylates RhoGDIα at Ser-174 and the phosphorylation of RhoGDIα is likely to induce the formation of a active RhoA-RhoGDIα complex. Our present results thus reveal a principal molecular mechanism underlying G(s)/cAMP-induced cross-talk with G(q)/G(13)/RhoA signaling.

The separate roles of endothelin receptors participate in remodeling of matrix metalloproteinase and connexin 43 of cardiac fibroblasts in maladaptive response to isoproterenol

Stress may affect gap junction connexin 43 and matrix metalloproteinase-2/9 (MMP-2/9) in cardiac fibroblasts, potentially contributing to worsening cardiac function and arrhythmias. Cardiac fibroblasts isolated from neonatal rat were incubated with isoproterenol at 3 × 10 − 7 M to mimic stress and were treated with either PD156707 or IRL-1038 (selective antagonists for endothelin A and B receptor respectively) and CPU0213 (a dual endothelin A/B receptor antagonist) at 1 × 10 − 8 M, 3 × 10 − 8 M or 1 × 10 − 7 M. RT-PCR and Western blotting were conducted. Upregulation of the two endothelin receptors, MMP-2/9 and NADPH oxidase subunits (p22phox and p47phox), and downregulation of connexin 43 in cardiac fibroblasts were found in the presence of isoproterenol and were attenuated by the selective blockers PD156707 and IRL-1038 in a dose-dependent manner. IRL-1038 was less effective. CPU0213 appeared to be more effective than the two selective blockers in blocking these changes. Changes in cardiac fibroblasts in response to isoproterenol mediated by upregulation of the endothelin–NADPH oxidase pathway may play a role in deteriorating cardiac function and arrhythmias. The endothelin A receptor has a major role, relative to the endothelin B receptor, in the remodeling of cardiac fibroblasts during isoproterenol stimulation. CPU0213, a dual endothelin receptor A/B blocker, seems to be more effective in normalizing these changes than do the selective endothelin receptor antagonists.

Mycoplasma alligatorisInfection Promotes CD95 (FasR) Expression and Apoptosis of Primary Cardiac Fibroblasts

Mycoplasma alligatoris causes acute lethal primary infection of susceptible hosts. A genome survey implicated sialidase and hyaluronidase, potential promoters of CD95-mediated eukaryotic cell death, as virulence factors of M. alligatoris. We used immunofluorescence imaging and flow cytometry to examine the effects of M. alligatoris infection in vitro on CD95 expression and apoptosis by alligator cardiac fibroblasts, a major cell type of a target organ of M. alligatoris infection in vivo. A uniform distribution of CD95 in primary cultured cardiac, skeletal muscle, and embryonic fibroblasts was demonstrated by using polyclonal antibodies against the N or C terminus of mouse or human CD95. Anti-CD95 antibodies reacted on Western blots of fibroblast lysates with a band with the predicted apparent molecular weight of CD95, but soluble CD95 was not detected in plasma from control or M. alligatoris-infected alligators. The proportion of CD95-gated cardiac fibroblasts increased threefold (P<0.01) 48 h after inoculation with M. alligatoris. Infection induced morphological changes in cardiac fibroblasts, including translocation of CD95 characteristic of apoptosis and an eightfold increase (P<0.16) in 5-bromo-2'-deoxyuridine (BrdU) incorporation measured in a terminal deoxynucleotide transferase dUTP nick end-labeling apoptosis assay. The proportion of BrdU-gated controls activated with agonistic immunoglobulin M against human CD95 also increased threefold (P<0.03 for muscle). Heat-inactivated M. alligatoris and sterile M. alligatoris-conditioned culture supernatant had no effect. This is the first report of a CD95 homolog in the class Reptilia and establishes a new model that can be used to test the direct bacterial interaction with upstream components of the CD95 signal transduction pathway.