Signaling In Cardiac Fibroblasts Research Articles

Phospholipase Cɛ Regulation of Cardiac Fibroblasts

Heart disease is one of the leading causes of death in the United States. Cardiac fibrosis is commonly associated with heart diseases, such as cardiac hypertrophy and myocardial infarctions. This is characterized as an excess deposition of extracellular matrix that results in a stiffening of the heart. Cardiac fibrosis can lead to arrhythmias, decreased diastolic function and eventually heart failure. Upon injury to the heart, cardiac fibroblasts transition to an activated myofibroblast state, characterized by the expression of α‐smooth muscle actin (αSMA). While the activation of fibroblasts is initially beneficial to the heart, chronic activation of fibroblasts leads to excessive deposition of collagen resulting in the development of fibrosis. Phospholipase Cɛ (PLCɛ) is an important signaling enzyme that is activated by small GTPases downstream of G‐protein coupled receptors (GPCRs) and receptor tyrosine kinases and is important in the cardiac myocyte for the development of cardiac hypertrophy. In this study we sought to elucidate the role of PLCɛ in the transition of cardiac fibroblasts to activated myofibroblasts. Using neonatal rat cardiac fibroblasts, we demonstrate that PLCɛ is highly expressed in fibroblasts compared to myocytes at the mRNA and protein level. Adenovirus encoding for a PLCɛ‐shRNA was used to knockdown PLCɛ in fibroblasts and the ability of the cells to transition to a myofibroblast was examined. Knocking down PLCɛ decreases the mRNA expression of pro‐fibrotic genes such as αSMA, collagen 1, and TGFβ indicating that PLCɛ can regulate pro‐fibrotic gene expression in cardiac fibroblasts. The transition to myofibroblasts induced by transforming growth factor‐β (TGFβ) is reduced in cells treated with PLCɛ‐shRNA compared to cells treated with a control shRNA as assessed by staining of αSMA. In addition, knocking down PLCɛ reduces the ability of the cardiac fibroblasts to respond to pro‐fibrotic Gq‐coupled GPCRs such as the angiotensin receptor and the protease‐activated receptor 1. These data indicate that PLCɛ has an important role in pro‐fibrotic signaling in cardiac fibroblasts and can regulate the myofibroblast transition. Future studies will focus on elucidating the molecular mechanism by which PLCɛ is involved in the pro‐fibrotic signaling response.Support or Funding InformationThis project is supported by grant number T32‐HL007853 from the NIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sacubitril/Valsartan Decreases Cardiac Fibrosis in Left Ventricle Pressure Overload by Restoring PKG Signaling in Cardiac Fibroblasts.

Background Heart failure (HF) is invariably accompanied by development of cardiac fibrosis, a form of scarring that increases muscular tissue rigidity and decreases cardiac contractility. Cardiac fibrosis arises from a pathological attempt to repair tissue damaged during maladaptive remodeling. Treatment options to block or reverse fibrosis have proven elusive. Neprilysin is an endopeptidase that degrades vasoactive peptides, including atrial natriuretic peptide. Thus, neprilysin inhibition reduces hypertension, ultimately limiting maladaptive cardiac remodeling. LCZ696, which consists of an angiotensin receptor blocker (valsartan [VAL]) and a neprilysin inhibitor (sacubitril [SAC]), was shown to be well tolerated and significantly reduced the risk of death and hospitalization in HF patients with reduced ejection fraction. We hypothesized that SAC/VAL directly inhibits fibroblast activation and development of pathological fibrosis. Methods and Results We used a mouse model of left ventricle pressure overload coupled to in vitro studies in primary mouse and human cardiac fibroblasts (CFs) to study the impact of SAC/VAL on CF activation and cardiac fibrosis. SAC/VAL significantly ameliorated pressure overload-induced cardiac fibrosis by blocking CF activation and proliferation, leading to functional improvement. Mechanistically, the beneficial impact of SAC/VAL at least partially stemmed from restoration of PKG (protein kinase G) signaling in HF patient-derived CF, which inhibited Rho activation associated with myofibroblast transition. Conclusions This study reveals that SAC/VAL acts directly on CF to prevent maladaptive cardiac fibrosis and dysfunction during pressure overload-induced hypertrophy and suggests that SAC/VAL should be evaluated as a direct antifibrotic therapeutic for conditions such as HF with preserved ejection fraction.

Macrophage Migration Inhibitory Factor (MIF) Expression Increases during Myocardial Infarction and Supports Pro-Inflammatory Signaling in Cardiac Fibroblasts.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine known to play a major role in inflammatory diseases such as myocardial infarction (MI), where its expression increases. Cardio-protective functions of MIF during ischemia have been reported. Recently, the structurally related MIF-2 was identified and similar effects are assumed. We wanted to further investigate the role of MIF and MIF-2 on inflammatory processes during MI. Therefore, we subjected mice to experimentally induced MI by coronary occlusion for one and five days. During the acute phase of MI, the gene expression of Mif was upregulated in the infarct zone, whereas Mif-2 was downregulated, suggesting a minor role of MIF-2. Simulating ischemic conditions or mechanical stress in vitro, we demonstrated that Mif expression was induced in resident cardiac cells. To investigate possible auto-/paracrine effects, cardiomyocytes and cardiac fibroblasts were individually treated with recombinant murine MIF, which in turn induced Mif expression and the expression of pro-inflammatory genes in cardiac fibroblasts. Cardiomyocytes did not respond to recombinant MIF with pro-inflammatory gene expression. While MIF stimulation alone did not change the expression of pro-fibrotic genes in cardiac fibroblasts, ischemia reduced their expression. Mimicking the increased MIF levels during MI, we exposed cardiac fibroblasts to simulated ischemia in the presence of MIF, which led to further reduced expression of pro-fibrotic genes. The presented data show that MIF was expressed by resident cardiac cells during MI. In vitro, Mif expression was induced by different external stimuli in cardiomyocytes and cardiac fibroblasts. Addition of recombinant MIF protein increased the expression of pro-inflammatory genes in cardiac fibroblasts including Mif expression itself. Thereby, cardiac fibroblasts may amplify Mif expression during ischemia promoting cardiomyocyte survival.

Myofibroblast β2 adrenergic signaling amplifies cardiac hypertrophy in mice

Abnormal β-adrenergic signaling plays a central role in human heart failure. In mice, chronic β-adrenergic receptor (βAR) stimulation elicits cardiac hypertrophy. It has been reported that cultured cardiac fibroblasts express βAR; however, the functional in vivo requirement of βAR signaling in cardiac fibroblasts during the development of cardiac hypertrophy remains elusive. β2AR null mice exhibited attenuated hypertrophic responses to chronic βAR stimulation upon continuous infusion of an agonist, isoprenaline (ISO), compared to those in wildtype controls, suggesting that β2AR activation in the heart induces pro-hypertrophic effects in mice. Since β2AR signaling is protective in cardiomyocytes, we focused on β2AR signaling in cardiac myofibroblasts. To determine whether β2AR signaling in myofibroblasts affects cardiac hypertrophy, we generated myofibroblast-specific transgenic mice (TG) with the catalytic subunit of protein kinase A (PKAcα) using Cre-loxP system. Myofibroblast-specific PKAcα overexpression resulted in enhanced heart weight normalized to body weight ratio, associated with an enlargement of cardiomyocytes at 12 weeks of age, indicating that myofibroblast-specific activation of PKA mediates cardiac hypertrophy in mice. Neonatal rat cardiomyocytes stimulated with conditioned media from TG cardiac fibroblasts likewise exhibited significantly more growth than those from controls. Thus, β2AR signaling in myofibroblasts plays a substantial role in ISO-induced cardiac hypertrophy, possibly due to a paracrine effect. β2AR signaling in cardiac myofibroblasts may represent a promising target for development of novel therapies for cardiac hypertrophy.

Mitoquinone ameliorates pressure overload-induced cardiac fibrosis and left ventricular dysfunction in mice

Increasing evidence indicates that mitochondrial-associated redox signaling contributes to the pathophysiology of heart failure (HF). The mitochondrial-targeted antioxidant, mitoquinone (MitoQ), is capable of modifying mitochondrial signaling and has shown beneficial effects on HF-dependent mitochondrial dysfunction. However, the potential therapeutic impact of MitoQ-based mitochondrial therapies for HF in response to pressure overload is reliant upon demonstration of improved cardiac contractile function and suppression of deleterious cardiac remodeling. Using a new (patho)physiologically relevant model of pressure overload-induced HF we tested the hypothesis that MitoQ is capable of ameliorating cardiac contractile dysfunction and suppressing fibrosis. To test this C57BL/6J mice were subjected to left ventricular (LV) pressure overload by ascending aortic constriction (AAC) followed by MitoQ treatment (2 µmol) for 7 consecutive days. Doppler echocardiography showed that AAC caused severe LV dysfunction and hypertrophic remodeling. MitoQ attenuated pressure overload-induced apoptosis, hypertrophic remodeling, fibrosis and LV dysfunction. Profibrogenic transforming growth factor-β1 (TGF-β1) and NADPH oxidase 4 (NOX4, a major modulator of fibrosis related redox signaling) expression increased markedly after AAC. MitoQ blunted TGF-β1 and NOX4 upregulation and the downstream ACC-dependent fibrotic gene expressions. In addition, MitoQ prevented Nrf2 downregulation and activation of TGF-β1-mediated profibrogenic signaling in cardiac fibroblasts (CF). Finally, MitoQ ameliorated the dysregulation of cardiac remodeling-associated long noncoding RNAs (lncRNAs) in AAC myocardium, phenylephrine-treated cardiomyocytes, and TGF-β1-treated CF. The present study demonstrates for the first time that MitoQ improves cardiac hypertrophic remodeling, fibrosis, LV dysfunction and dysregulation of lncRNAs in pressure overload hearts, by inhibiting the interplay between TGF-β1 and mitochondrial associated redox signaling.

Transient Receptor Potential Canonical 3 and Nuclear Factor of Activated T Cells C3 Signaling Pathway Critically Regulates Myocardial Fibrosis.

Cardiac fibroblasts (CFs) are emerging as major contributors to myocardial fibrosis (MF), a final common pathway of many etiologies of heart disease. Here, we studied the functional relevance of transient receptor potential canonical 3 (TRPC3) channels and nuclear factor of activated T cells c3 (NFATc3) signaling in rodent and human ventricular CFs, and whether their modulation would limit MF. A positive feedback loop between TRPC3 and NFATc3 drove a rat ventricular CF fibrotic phenotype. In these cells, polyphenols (extract of grape pomace polyphenol [P.E.]) decreased basal and angiotensin II-mediated Ca2+ entries through a direct modulation of TRPC3 channels and subsequently NFATc3 signaling, abrogating myofibroblast differentiation, fibrosis and inflammation, as well as an oxidative stress-associated phenotype. N(ω)-nitro-l-arginine methyl ester (l-NAME) hypertensive rats developed coronary perivascular, sub-epicardial, and interstitial fibrosis with induction of embryonic epicardial progenitor transcription factors in activated CFs. P.E. treatment reduced ventricular CF activation by modulating the TRPC3-NFATc3 pathway, and it ameliorated echocardiographic parameters, cardiac stress markers, and MF in l-NAME hypertensive rats independently of blood pressure regulation. Further, genetic deletion (TRPC3-/-) and pharmacological channel blockade with N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-benzenesulfonamide (Pyr10) blunted ventricular CF activation and MF in l-NAME hypertensive mice. Finally, TRPC3 was present in human ventricular CFs and upregulated in MF, whereas pharmacological modulation of TRPC3-NFATc3 decreased proliferation and collagen secretion. Innovation and Conclusion: We demonstrate that TRPC3-NFATc3 signaling is modulated by P.E. and critically regulates ventricular CF phenotype and MF. These findings strongly argue for P.E., through TRPC3 targeting, as potential and interesting therapeutics for MF management.

CaMKIIδ interacts directly with IKKβ and modulates NF-κB signalling in adult cardiac fibroblasts

Calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) acts as a molecular switch regulating cardiovascular Ca2+ handling and contractility in health and disease. Activation of CaMKIIδ is also known to regulate cardiovascular inflammation and is reported to be required for pro-inflammatory NF-κB signalling. In this study the aim was to characterise how CaMKIIδ interacts with and modulates NF-κB signalling and whether this interaction exists in non-contractile cells of the heart. Recombinant or purified CaMKIIδ and the individual inhibitory -κB kinase (IKK) proteins of the NF-κB signalling pathway were used in autoradiography and Surface Plasmon Resonance (SPR) to explore potential interactions between both components. Primary adult rat cardiac fibroblasts were then used to study the effects of selective CaMKII inhibition on pharmacologically-induced NF-κB activation as well as interaction between CaMKII and specific IKK isoforms in a cardiac cellular setting. Autoradiography analysis suggested that CaMKIIδ phosphorylated IKKβ but not IKKα. SPR analysis further supported a direct interaction between CaMKIIδ and IKKβ but not between CaMKIIδ and IKKα or IKKγ. CaMKIIδ regulation of IκΒα degradation was explored in adult cardiac fibroblasts exposed to pharmacological stimulation. Cells were stimulated with agonist in the presence or absence of a CaMKII inhibitor, autocamtide inhibitory peptide (AIP). Selective inhibition of CaMKII resulted in reduced NF-κB activation, as measured by agonist-stimulated IκBα degradation. Importantly, and in agreement with the recombinant protein work, an interaction between CaMKII and IKKβ was evident following Proximity Ligation Assays in adult cardiac fibroblasts. This study provides new evidence supporting direct interaction between CaMKIIδ and IKKβ in pro-inflammatory signalling in cardiac fibroblasts and could represent a feature that may be exploited for therapeutic benefit.

Scoparone attenuates angiotensin II-induced extracellular matrix remodeling in cardiac fibroblasts

Scoparone is a biologically active constituent isolated from Artemisia capillaris and possesses a variety of pharmacological activities, such as anti-inflammatory, anti-tumor, anti-allergic and anti-cardiovascular activities. However, there are no studies focusing on the effects of scoparone against cardiac fibrosis. Therefore, the aim of this study was to investigate the effects of scoparone on Angiotensin II (Ang II)-induced extracellular matrix (ECM) remodeling and its possible mechanism in cardiac fibroblasts (CFs). Our results demonstrated that scoparone effectively attenuated CFs proliferation in Ang II-stimulated CFs. Scoparone also prevented the differentiation of CFs to myofibroblasts and ECM proteins (type I collagen and fibronectin) expression in Ang II-stimulated CFs. Furthermore, scoparone prevented Ang II-induced the activation of TGF-β1/Smad signalling in CFs. Taken together, these studies indicated that scoparone attenuated Ang II-induced ECM remodeling in CFs, at least in part, by inhibiting TGF-β1/Smad signalling. These findings suggest that scoparone may be used a novel therapeutic agent against cardiac fibrosis.

Tanshinone IIA Inhibits High Glucose-Induced Collagen Synthesis via Nuclear Factor Erythroid 2-Related Factor 2 in Cardiac Fibroblasts

Background/Aims: Diabetes is associated with increased incidence of myocardial dysfunction, which is partly characterized by interstitial and perivascular fibrosis. Cardiac fibroblasts have been identified as an important participant in the development of cardiac fibrosis. Exposure of cultured cardiac fibroblasts to high glucose resulted in increased collagen synthesis. Tanshinone IIA can alleviate the ventricular fibrosis that develops in a number of different experimental conditions. However, whether tanshinone IIA can prevent high glucose-induced collagen synthesis in cardiac fibroblasts remains unknown. The aim of this study was to evaluate the effects of tanshinone IIA on high glucose-induced collagen synthesis in cardiac fibroblasts. Methods: Rat cardiac fibroblasts were cultured in high glucose (25 mM) media in the absence or presence of tanshinone IIA and the changes in collagen synthesis, transforming growth factor-β1 (TGF-β1) production and related signaling molecules were assessed by ³H-proline incorporation, quantitative polymerase chain reaction, enzyme linked immunosorbent assay, and Western blotting. Results: The results indicate cardiac fibroblasts exposed to high glucose condition show increased cell proliferation and collagen synthesis and these effects were abolished by tanshinone IIA treatment. Furthermore, the inhibitory effect of tanshinone IIA on high glucose induced cell proliferation and collagen synthesis may be associated with its activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and the inhibition of TGF-β1 production and Smad2/3 phosphorylation. Conclusion: In summary, our results highlights the critical role tanshinone IIA plays as an antioxidant in attenuating high glucose-mediated collagen synthesis through inhibiting TGF-β1/Smad signaling in cardiac fibroblasts which provide a mechanistic basis for the clinical application of tanshinone IIA in the treating diabetic-related cardiac fibrosis.

Loss of \u03b2-catenin in resident cardiac fibroblasts attenuates fibrosis induced by pressure overload in mice

Cardiac fibrosis is characterized by excessive extracellular matrix deposition that contributes to compromised cardiac function and potentially heart failure. Cardiac pressure overload resulting from trans-aortic constriction in mice leads to cardiac fibrosis and increased Wnt/β-catenin signaling in cardiac fibroblasts. Here, we conditionally induce β-catenin loss of function in resident cardiac fibroblasts using Tcf21MerCreMer or in activated cardiac fibroblasts using periostin (Postn)MerCreMer. We show that β-catenin loss of function in cardiac fibroblasts after trans-aortic constriction significantly preserves cardiac function, and reduces interstitial fibrosis but does not alter the numbers of activated or differentiated cardiac fibroblasts in vivo. However, β-catenin is specifically required in resident cardiac fibroblasts for fibrotic excessive extracellular matrix gene expression and binds Col3a1 and Postn gene sequences in cultured cardiac fibroblasts after induction of Wnt signaling. Moreover, cardiomyocyte hypertrophy is blunted with cardiac fibroblast-specific loss of β-catenin after trans-aortic constriction in vivo. Thus, Wnt/β-catenin signaling in resident cardiac fibroblasts is required for excessive extracellular matrix gene expression and collagen deposition after trans-aortic constriction.

Augmented sphingosine 1 phosphate receptor-1 signaling in cardiac fibroblasts induces cardiac hypertrophy and fibrosis through angiotensin II and interleukin-6.

Background: Cardiac fibroblasts, together with cardiomyocytes, occupy the majority of cells in the myocardium and are involved in myocardial remodeling. The lysophospholipid mediator sphigosine-1-phosphate (S1P) regulates functions of cardiovascular cells through multiple receptors including S1PR1–S1PR3. S1PR1 but not other S1P receptors was upregulated in angiotensin II-induced hypertrophic hearts. Therefore, we investigated a role of S1PR1 in fibroblasts for cardiac remodeling by employing transgenic mice that overexpressed S1PR1 under the control of α-smooth muscle actin promoter. In S1PR1-transgenic mouse heart, fibroblasts and/or myofibroblasts were hyperplastic, and those cells as well as vascular smooth muscle cells overexpressed S1PR1. Transgenic mice developed bi-ventricular hypertrophy by 12-week-old and diffuse interstitial fibrosis by 24-week-old without hemodynamic stress. Cardiac remodeling in transgenic mice was associated with greater ERK phosphorylation, upregulation of fetal genes, and systolic dysfunction. Transgenic mouse heart showed increased mRNA expression of angiotensin-converting enzyme and interleukin-6 (IL-6). Isolated fibroblasts from transgenic mice exhibited enhanced generation of angiotensin II, which in turn stimulated IL-6 release. Either an AT1 blocker or angiotensin-converting enzyme inhibitor prevented development of cardiac hypertrophy and fibrosis, systolic dysfunction and increased IL-6 expression in transgenic mice. Finally, administration of anti-IL-6 antibody abolished an increase in tyrosine phosphorylation of STAT3, a major signaling molecule downstream of IL-6, in the transgenic mouse heart and prevented development of cardiac hypertrophy in transgenic mice. These results demonstrate a promoting role of S1PR1 in cardiac fibroblasts for cardiac remodeling, in which angiotensin II—AT1 and IL-6 are involved.

IL-21 promotes myocardial ischaemia/reperfusion injury through the modulation of neutrophil infiltration.

The immune system plays an important role in driving the acute inflammatory response following myocardial ischaemia/reperfusion injury (MIRI). IL-21 is a pleiotropic cytokine with multiple immunomodulatory effects, but its role in MIRI is not known. Myocardial injury, neutrophil infiltration and the expression of neutrophil chemokines KC (CXCL1) and MIP-2 (CXCL2) were studied in a mouse model of MIRI. Effects of IL-21 on the expression of KC and MIP-2 in neonatal mouse cardiomyocytes (CMs) and cardiac fibroblasts (CFs) were determined by real-time PCR and ELISA. The signalling mechanisms underlying these effects were explored by western blot analysis. IL-21 was elevated within the acute phase of murine MIRI. Neutralization of IL-21 attenuated myocardial injury, as illustrated by reduced infarct size, decreased cardiac troponin T levels and improved cardiac function, whereas exogenous IL-21 administration exerted opposite effects. IL-21 increased the infiltration of neutrophils and increased the expression of KC and MIP-2 in myocardial tissue following MIRI. Moreover, neutrophil depletion attenuated the IL-21-induced myocardial injury. Mechanistically, IL-21 increased the production of KC and MIP-2 in neonatal CMs and CFs, and enhanced neutrophil migration, as revealed by the migration assay. Furthermore, we demonstrated that this IL-21-mediated increase in chemokine expression involved the activation of Akt/NF-κB signalling in CMs and p38 MAPK/NF-κB signalling in CFs. Our data provide novel evidence that IL-21 plays a pathogenic role in MIRI, most likely by promoting cardiac neutrophil infiltration. Therefore, targeting IL-21 may have therapeutic potential as a treatment for MIRI. This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

A peptide vaccine targeting angiotensin II attenuates the cardiac dysfunction induced by myocardial infarction

A peptide vaccine targeting angiotensin II (Ang II) was recently developed as a novel treatment for hypertension to resolve the problem of noncompliance with pharmacotherapy. Ang II plays a crucial role in the pathogenesis of cardiac remodeling after myocardial infarction (MI), which causes heart failure. In the present study, we examined whether the Ang II vaccine is effective in preventing heart failure. The injection of the Ang II vaccine in a rat model of MI attenuated cardiac dysfunction in association with an elevation in the serum anti-Ang II antibody titer. Furthermore, any detrimental effects of the Ang II vaccine were not observed in the rats that underwent sham operations. Treatment with immunized serum from Ang II vaccine-injected rats significantly suppressed post-MI cardiac dysfunction in MI rats and Ang II-induced remodeling-associated signaling in cardiac fibroblasts. Thus, our present study demonstrates that the Ang II vaccine may provide a promising novel therapeutic strategy for preventing heart failure.

Effect of α7 nicotinic acetylcholine receptor activation on cardiac fibroblasts: a mechanism underlying RV fibrosis associated with cigarette smoke exposure.

Right ventricular (RV) dysfunction is associated with numerous smoking-related illnesses, including chronic obstructive pulmonary disease (COPD), in which it is present even in the absence of pulmonary hypertension. It is unknown whether exposure to cigarette smoke (CS) has direct effects on RV function and cardiac fibroblast (CF) proliferation or collagen synthesis. In this study, we evaluated cardiac function and fibrosis in mice exposed to CS and determined mechanisms of smoke-induced changes in CF signaling and fibrosis. AKR mice were exposed to CS for 6 wk followed by echocardiography and evaluation of cardiac hypertrophy, collagen content, and pulmonary muscularization. Proliferation and collagen content were evaluated in primary isolated rat CFs exposed to CS extract (CSE) or nicotine. Markers of cell proliferation, fibrosis, and proliferative signaling were determined by immunoblot or Sircol collagen assay. Mice exposed to CS had significantly decreased RV function, as determined by tricuspid annular plane systolic excursion. There were no changes in left ventricular parameters. RV collagen content was significantly elevated, but there was no change in RV hypertrophy or pulmonary vascular muscularization. CSE directly increased CF proliferation and collagen content in CF. Nicotine alone reproduced these effects. CSE and nicotine-induced fibroblast proliferation and collagen content were mediated through α7 nicotinic acetylcholine receptors and were dependent on PKC-α, PKC-δ, and reduced p38-MAPK phosphorylation. CS and nicotine have direct effects on CFs to induce proliferation and fibrosis, which may negatively affect right heart function.

Myocyte-Derived Hsp90 Modulates Collagen Upregulation via Biphasic Activation of STAT-3 in Fibroblasts during Cardiac Hypertrophy.

Signal transducer and activator of transcription 3 (STAT-3)-mediated signaling in relation to upregulated collagen expression in fibroblasts during cardiac hypertrophy is well defined. Our recent findings have identified heat shock protein 90 (Hsp90) to be a critical modulator of fibrotic signaling in cardiac fibroblasts in this disease milieu. The present study was therefore intended to analyze the role of Hsp90 in the STAT-3-mediated collagen upregulation process. Our data revealed a significant difference between in vivo and in vitro results, pointing to a possible involvement of myocyte-fibroblast cross talk in this process. Cardiomyocyte-targeted knockdown of Hsp90 in rats (Rattus norvegicus) in which the renal artery was ligated showed downregulated collagen synthesis. Furthermore, the results obtained with cardiac fibroblasts conditioned with Hsp90-inhibited hypertrophied myocyte supernatant pointed toward cardiomyocytes' role in the regulation of collagen expression in fibroblasts during hypertrophy. Our study also revealed a novel signaling mechanism where myocyte-derived Hsp90 orchestrates not only p65-mediated interleukin-6 (IL-6) synthesis but also its release in exosomal vesicles. Such myocyte-derived exosomes and myocyte-secreted IL-6 are responsible in unison for the biphasic activation of STAT-3 signaling in cardiac fibroblasts that culminates in excess collagen synthesis, leading to severely compromised cardiac function during cardiac hypertrophy.

Mitochondrial Ca2+ Uptake and Superoxide Generation Regulates Angiotensin II-Induced Proliferation in Neonatal Cardiac Fibroblasts

Cardiac fibroblasts (CFs) are one o the most abundant cell types in the heart and play key roles in regulating myocardial physiological function and pathophysiological remodeling especially for the cardiac fibrosis. The levels of Angiotensin II (AT-II) are increased in the remodeling heart and Angiotensin signaling participates in pathological CF proliferation. It has been shown that CF proliferation may occur via the increased levels of cellular reactive oxygen species (ROS), but the detailed signal transduction remains unclear. We previously reported that the enhancement of mitochondrial Ca2+ uptake by mitochondrial Ca2+ uniporter (MCU) induces mitochondrial superoxide (mtSO) generation in cardiac myofibroblast cell line H9C2 cells. Therefore, we hypothesize that Ang-II stimulation enhances mitochondrial Ca2+-induced mtSO generation in primary CFs, which can activate ROS-dependent proliferation signaling in primary CFs. First, we confirmed that AT-II (≥1 μM) stimulation induces significant mitochondrial Ca2+ uptake assess by mitochondria-targeted Ca2+ biosensor in response to the Ca2+ release from the endoplasmic reticulum in neonatal rat CFs (NCF). In addition, AT-II stimulation increases the mtSO levels detected by a mtSO indicator MitoSOX Red. We also confirmed that AT-II application activates proliferative pathway, including ERK1/2, p38 and JNK1/2 in time-dependent manner, which was abolished by losartan pretreatment. Lastly, pretreatment of a mitochondria-targeted antioxidant, Mito-tempo significantly inhibited AT-II-mediated activation of the mtSO production as well as proliferative pathway without changing the AT-II-induced the mitochondrial Ca2+ uptake profile. Our results indicate that mtSO generation induced by mitochondrial Ca2+ accumulation via MCU serves as an important regulator for the Ang II-induced proliferation in CFs.

Understanding the Role of Stiffness in Pathological Cardiac Fibroblast Signaling

Cardiac fibrosis is characterized by extracellular matrix buildup and tissue stiffening. Fibroblasts in the heart respond to pathological cues by activating to proliferate, upregulate expression of alpha smooth muscle actin, synthesize extracellular matrix protein, and secrete biochemical cues to surrounding cells. The cues that trigger this transformation remain incompletely resolved; however, stiffness appears to play a critical role in this process. In vitro experiments using hydrogels with different stiffnesses show that fibroblasts activate when cultured on stiff hydrogels while they remain unactivated on soft hydrogels as they do in the diseased and healthy heart, respectively. Understanding fibroblast activation through mechanosensitive signaling will provide new therapeutic targets for fibrosis treatment.

Palmitate promotes inflammatory responses and cellular senescence in cardiac fibroblasts

Palmitate triggers inflammatory responses in several cell types, but its effects on cardiac fibroblasts are at present unknown. The aims of the study were to (1) assess the potential of palmitate to promote inflammatory signaling in cardiac fibroblasts through TLR4 and the NLRP3 inflammasome and (2) characterize the cellular phenotype of cardiac fibroblasts exposed to palmitate. We examined whether palmitate induces inflammatory responses in cardiac fibroblasts from WT, NLRP3−/− and ASC−/−mice (C57BL/6 background). Exposure to palmitate caused production of TNF, IL-6 and CXCL2 via TLR4 activation. NLRP3 inflammasomes are activated in a two-step manner. Whereas palmitate did not prime the NLRP3 inflammasome, it induced activation in LPS-primed cardiac fibroblasts as indicated by IL-1β, IL-18 production and NLRP3-ASC co-localization. Palmitate-induced NLRP3 inflammasome activation in LPS-primed cardiac fibroblasts was associated with reduced AMPK activity, mitochondrial reactive oxygen species production and mitochondrial dysfunction. The cardiac fibroblast phenotype caused by palmitate, in an LPS and NLRP3 independent manner, was characterized by decreased cellular proliferation, contractility, collagen and MMP-2 expression, as well as increased senescence-associated β-galactosidase activity, and consistent with a state of cellular senescence. This study establishes that in vitro palmitate exposure of cardiac fibroblasts provides inflammatory responses via TLR4 and NLRP3 inflammasome activation. Palmitate also modulates cardiac fibroblast functionality, in a NLRP3 independent manner, resulting in a phenotype related to cellular senescence. These effects of palmitate could be of importance for myocardial dysfunction in obese and diabetic patients.

Abstract 12745: Interleukin-4 Promotes Procollagen Type-I Alpha 1 Gene Expression via Activator Protein 1 Activation in Cardiac Fibroblasts

Interleukin-4 (IL-4) activates the signal transducer and activator of transcription 6 signaling pathway, resulting in the upregulation of procollagen genes and promotion of collagen production in cardiac fibroblasts (CFs). In addition, transcription factors may participate in the transcriptional activation of collagen genes. We hypothesize that IL-4 activates activator protein 1 (AP-1) via c-Jun phosphorylation, promoting transcriptional activation of the procollagen type-I alpha 1 (Col1α1) gene and collagen type-I expression in CFs. Primary mouse CFs were used. Western blot analyses revealed that IL-4 (10 ng/ml) increased phosphorylation of c-Jun, a subunit of AP-1. Additionally, our experiments utilizing electrophoretic mobility shift assay (EMSA) revealed a significant increase in DNA binding activity for AP-1 in IL-4-treated CFs compared to control cells (1.00±0.07 to 2.14±0.38 fold change versus control, p <0.05). Here we aimed to determine whether IL-4-activated AP-1 participates in transcriptional activation of the Col1α1 gene leading to increased collagen type-I expression in CFs. To that end, we blocked AP-1 with shRNA lentiviral particles specific for c-Jun in CFs, detected Col1α1 mRNA by qRT-PCR, and measured collagen type-I by Western blot. We found that IL-4 significantly induced Col1α1 mRNA expression after incubation with control shRNA-transduced cells for 6 hours (1.01±0.01 to 1.52±0.07 fold change versus control, p <0.005), and it increased collagen type-I expression after 24 hours of incubation (1.00±0.01 to 1.95±0.12 fold change versus control, p <0.005). In contrast, IL-4 did not increase mRNA levels of Col1α1 (1.09±0.02 to 0.98±0.03, p >0.05) and the collagen type-I (0.95±0.05 to 1.01±0.10, p >0.05) in c-Jun shRNA-transfected cells. Our data demonstrate that IL-4-activated AP-1, an ubiquitous transcription factor containing phosphorylated c-Jun, enhanced Col1α1 gene expression and collagen type-I expression in CFs. Data generated from this study provides a novel molecular basis for potential treatment of cardiac fibrosis by targeting IL-4-induced signaling in CFs.

Basic Signaling in Cardiac Fibroblasts.

Cardiac fibroblasts are commonly known as supporting cells of the cardiac network and exert many essential functions that are fundamental for normal cardiac growth as well as for cardiac remodeling process during pathological conditions. This review focuses on the roles of cardiac fibroblasts in the formation and regulation of the extracellular matrix components, and in maintaining structural, biochemical and mechanical properties of the heart. Additionally, though considered as non-excitable cells, we review the functional expression in cardiac fibroblasts of a wide variety of transmembrane ion channels which activity may contribute to key regulation of cardiac physiological processes. All together, cardiac fibroblasts which actively participate to fundamental regulation of cardiac physiology and physiopathology processes may represent pertinent targets for pharmacological approaches of cardiac diseases and lead to new tracks of therapeutic strategies. J. Cell. Physiol. 232: 725-730, 2017. © 2016 Wiley Periodicals, Inc.