Adult Rat Cardiac Fibroblasts Research Articles

Culturing of Cardiac Fibroblasts in Engineered Heart Matrix Reduces Myofibroblast Differentiation but Maintains Their Response to Cyclic Stretch and Transforming Growth Factor β1.

Isolation and culturing of cardiac fibroblasts (CF) induces rapid differentiation toward a myofibroblast phenotype, which is partly mediated by the high substrate stiffness of the culture plates. In the present study, a 3D model of Engineered Heart Matrix (EHM) of physiological stiffness (Youngs modulus ~15 kPa) was developed using primary adult rat CF and a natural hydrogel collagen type 1 matrix. CF were equally distributed, viable and quiescent for at least 13 days in EHM and the baseline gene expression of myofibroblast-markers alfa-smooth muscle actin (Acta2), and connective tissue growth factor (Ctgf) was significantly lower, compared to CF cultured in 2D monolayers. CF baseline gene expression of transforming growth factor-beta1 (Tgfβ1) and brain natriuretic peptide (Nppb) was higher in EHM-fibers compared to the monolayers. EHM stimulation by 10% cyclic stretch (1 Hz) increased the gene expression of Nppb (3.0-fold), Ctgf (2.1-fold) and Tgfβ1 (2.3-fold) after 24 h. Stimulation of EHM with TGFβ1 (1 ng/mL, 24 h) induced Tgfβ1 (1.6-fold) and Ctgf (1.6-fold). In conclusion, culturing CF in EHM of physiological stiffness reduced myofibroblast marker gene expression, while the CF response to stretch or TGFβ1 was maintained, indicating that our novel EHM structure provides a good physiological model to study CF function and myofibroblast differentiation.

Abstract P1071: Concurrent Stat1 And Stat3 Signaling Modulates Tlr4/nfkb-mediated Immunomodulatory And Antifibrotic Effects Of Cardiomyocyte-damaged Released S100a1 Protein In Cardiac Fibroblasts

Background: We previously showed that S100A1 released from damaged cardiomyocytes can accelerate post-myocardial infarction in vivo healing. The action of S100A1 as a damage-associated molecular pattern seemed to occur via an immunomodulatory (e.g., IL10 increase) and antifibrotic impact (e.g., COL1A1 and ACTA2 downregulation) in cardiac fibroblasts (CFs) via a TRL4/NKFB pathway. The signaling network in CFs conveying this effect has not been characterized yet. Methods and Results: We performed a 48h time-resolved transcriptome analysis on cultured adult rat CF stimulated with rec. human S100A1 protein (10 -6 M). Bioinformatic GSEA yielded an instant and almost exclusive significant upregulation of the chemokine activity GO-term with a delayed suppression of ECM genes. A transcription factor enrichment analysis for the differentially regulated transcripts indicated an immediate activation of NFKB/RELA with a concurrent temporary STAT1 but lasting STAT3 signaling. A secretome LC-MS/MS-based analysis confirmed the shift of S100A1-treated CFs to mostly chemokine secreting cells. With CCL2 as most secreted chemokine and COL1A1 and ACTA2 as established antifibrotic marker genes for released S100A1, we used a RT-PCR readout for these genes in response to siRNA-based knock-down (KD) of STAT1 and STAT3 in S100A1-treated CFs, while TLR4 KD was used to abrogate NFKB/RELA signaling due to known mechanisms (n=10 each group). S100A1 treatment upregulated CCL2 (16-fold*) and downregulated COL1A1 (3.1-fold*) and ACTA2 (2.9-fold*) mRNA expression vs control (*P<0.01) that was blunted by TLR4 KD. STAT3 KD doubled the CCL2 mRNA increase (32.9-fold*), attenuated COL1A1 mRNA downregulation by S100A1 treatment to 2-fold* but left ACTA2 downregulation unaffected vs contr. (*P<0.05). Conversely, STAT1 KD completely blocked the S100A1 mediated downregulation of ACTA2 but neither impacted S100A1-mediated changes in CCL2 nor COL1A1. Conclusion: Our study identifies concurrent STAT1 and STAT3 activation as a modulator of S100A1-mediated TLR4-NFKB signaling in CFs. As S100A1-activated CFs express e.g., IL-10, we speculate that IL-gp130 trans signaling may convey this effect guiding further studies on S100A1’s extracellular effects in the diseased heart.

P63 silencing induces epigenetic modulation to enhance human cardiac fibroblast to cardiomyocyte-like differentiation

Direct cell reprogramming represents a promising new myocardial regeneration strategy involving in situ transdifferentiation of cardiac fibroblasts into induced cardiomyocytes. Adult human cells are relatively resistant to reprogramming, however, likely because of epigenetic restraints on reprogramming gene activation. We hypothesized that modulation of the epigenetic regulator gene p63 could improve the efficiency of human cell cardio-differentiation. qRT-PCR analysis demonstrated significantly increased expression of a panel of cardiomyocyte marker genes in neonatal rat and adult rat and human cardiac fibroblasts treated with p63 shRNA (shp63) and the cardio-differentiation factors Hand2/Myocardin (H/M) versus treatment with Gata4, Mef2c and Tbx5 (GMT) with or without shp63 (p < 0.001). FACS analysis demonstrated that shp63+ H/M treatment of human cardiac fibroblasts significantly increased the percentage of cells expressing the cardiomyocyte marker cTnT compared to GMT treatment with or without shp63 (14.8% ± 1.4% versus 4.3% ± 1.1% and 3.1% ± 0.98%, respectively; p < 0.001). We further demonstrated that overexpression of the p63—transactivation inhibitory domain (TID) interferes with the physical interaction of p63 with the epigenetic regulator HDAC1 and that human cardiac fibroblasts treated with p63-TID+ H/M demonstrate increased cardiomyocyte marker gene expression compared to cells treated with shp63+ H/M (p < 0.05). Whereas human cardiac fibroblasts treated with GMT alone failed to contract in co-culture experiments, human cardiac fibroblasts treated with shp63+ HM or p63-TID+ H/M demonstrated calcium transients upon electrical stimulation and contractility synchronous with surrounding neonatal cardiomyocytes. These findings demonstrate that p63 silencing provides enhanced rat and human cardiac fibroblast transdifferentiation into induced cardiomyocytes compared to a standard reprogramming strategy. p63-TID overexpression may be a useful reprogramming strategy for overcoming epigenetic barriers to human fibroblast cardio-differentiation.

Sirt6 activation blocks LPS induced expression of inflammatory cytokines in cardiac cells

Sepsis induced myocardial dysfunction is a common complication that has been linked to an increased mortality. The activation of NFkB pathway and excessive production of pro‐inflammatory cytokines is considered to be one of the most important signaling pathway involved in the pathogenesis of sepsis. Earlier studies have shown that Sirt6, a member of class III of histone deacetylases, attenuates hyper‐activation of NFkB signaling that contributes to inflammation and the aging process. In the present study we tested the hypothesis whether pharmacological activation of Sirt6 can prevent LPS induced myocardial inflammation. Primary cultures of adult rat cardiac fibroblasts were treated with UBCS039, an allosteric activator of Sirt6, for 48 hours. The results showed a dose‐dependent increase in steady‐state levels of Sirt6 as well as deacetylation of its target H3K9 by Western analysis. A similar effect of UBCS039 treatment was observed in H9C2 cardiac myocytes. We also tested effects of UBCS039 on LPS induced NFkB activation and cytokine production in these cells. Our data showed that pretreatment of cardiac fibroblasts with UBCS039 for 48 hrs significantly reduced LPS (100ng/ml)‐induced phosphorylated levels of NFkB (pNFkB). By real‐time PCR analysis we found that LPS led to increased expression (4‐9 fold) of mRNA of IL‐6, IL‐1b, TLR4 and CD‐38 in cardiac fibroblasts within 6h of treatment. This effect of LPS in cardiac fibroblasts was completely blocked when cells were pretreated with UBCS039. These data thus indicated that pharmacological activation of Sirt6 is capable of blocking LPS induced inflammatory response in cardiac cells, and has the potential to alleviate sepsis induced myocardial dysfunction and related health consequences.

Long Non‐coding RNAs Mediate Heart Failure with Preserved Ejection Fraction Associated with Thyroid Dysfunction

IntroductionHeart failure with preserved ejection fraction (HEpEF) carries significantly high morbidity and mortality. HFpEF is associated with preserved contractile function and can present with diastolic dysfunction, cardiac fibrosis and hypothyroidism (low levels of thyroid hormones, THs). We previously demonstrated safe, cardioprotective effects with treatment of active form of THs in multiple rodent models of cardiovascular disorders. Long‐noncoding ribonucleic acids (lncRNAs; &gt;200 nucleotides long) account for large majority of the genome with significant transcriptional potential. However, their role in HFpEF associated with TH dysfunction is unclear. We hypothesized that lncRNAs act as mediators in development of hypothyroid HFpEF.MethodsHearts were isolated from obese ZSF1 rats that presented with HFpEF phenotype. Wild type and ZSF1 lean rats served as controls. Cardiac ultrasound and morphometric analyses were performed. RNA was isolated from left ventricular (LV) tissues and studied using Agilent LncRNA Microarray platform and analyzed using Feature Extraction and GeneSpring. Fibroblasts were isolated from wild type adult rat hearts and treated with TH receptor inhibitor. Real‐time quantitative polymerase chain reaction was used to study lncRNA expression.ResultsStudies revealed that total thyroxine TH levels significantly decreased in five‐six‐month old (early; p&lt;0.05) ZSF1 obese rats compared to ZSF1 lean controls (45% decrease) and wild type controls (42% decrease), and this remained low at 13 months (late; p&lt;0.05). Total tri‐iodothyronine TH levels also followed similar pattern in the early time‐point (37%‐42% decrease). ZSF1 obese rats showed increased normalized heart weight, LV weight and liver weight indicating cardiac hypertrophy and development of HF. In early obese rats, echocardiography showed preserved LV EF and significantly elevated E/E’ indicating diastolic dysfunction (p&lt;0.05). LncRNA Microarray analysis including unsupervised hierarchical clustering showed that several unique lncRNAs were significantly altered (p&lt;0.05; &gt;2‐fold change) in obese LVs compared to the lean or wild type control counterparts and select lncRNA expression was validated with qPCR. Furthermore, isolated wild type adult rat cardiac fibroblasts subjected to TH receptor inhibition (mimicking hypothyroidism) in vitro showed significant increase in lncRNA expression that was also increased in obese ZSF1 hearts presenting with hypothyroidism (p&lt;0.05).ConclusionsThese results indicate that lncRNAs may play important roles in in HFpEF pathogenesis associated with hypothyroidism. These studies will serve as significant step in identification of key lncRNAs in cardiac‐thyroid axis offering potentially valuable translational opportunities.

Piezo1 Mechanosensitive Ion Channel Mediates Stretch-Induced Nppb Expression in Adult Rat Cardiac Fibroblasts.

In response to stretch, cardiac tissue produces natriuretic peptides, which have been suggested to have beneficial effects in heart failure patients. In the present study, we explored the mechanism of stretch-induced brain natriuretic peptide (Nppb) expression in cardiac fibroblasts. Primary adult rat cardiac fibroblasts subjected to 4 h or 24 h of cyclic stretch (10% 1 Hz) showed a 6.6-fold or 3.2-fold (p < 0.05) increased mRNA expression of Nppb, as well as induction of genes related to myofibroblast differentiation. Moreover, BNP protein secretion was upregulated 5.3-fold in stretched cardiac fibroblasts. Recombinant BNP inhibited TGFβ1-induced Acta2 expression. Nppb expression was >20-fold higher in cardiomyocytes than in cardiac fibroblasts, indicating that cardiac fibroblasts were not the main source of Nppb in the healthy heart. Yoda1, an agonist of the Piezo1 mechanosensitive ion channel, increased Nppb expression 2.1-fold (p < 0.05) and significantly induced other extracellular matrix (ECM) remodeling genes. Silencing of Piezo1 reduced the stretch-induced Nppb and Tgfb1 expression in cardiac fibroblasts. In conclusion, our study identifies Piezo1 as mediator of stretch-induced Nppb expression, as well as other remodeling genes, in cardiac fibroblasts.

Sunitinib and Imatinib Display Differential Cardiotoxicity in Adult Rat Cardiac Fibroblasts That Involves a Role for Calcium/Calmodulin Dependent Protein Kinase II.

Background: Tyrosine kinase inhibitors (TKIs) have dramatically improved cancer treatment but are known to cause cardiotoxicity. The pathophysiological consequences of TKI therapy are likely to manifest across different cell types of the heart, yet there is little understanding of the differential adverse cellular effects. Cardiac fibroblasts (CFs) play a pivotal role in the repair and remodeling of the heart following insult or injury, yet their involvement in anti-cancer drug induced cardiotoxicity has been largely overlooked. Here, we examine the direct effects of sunitinib malate and imatinib mesylate on adult rat CF viability, Ca2+ handling and mitochondrial function that may contribute to TKI-induced cardiotoxicity. In particular, we investigate whether Ca2+/calmodulin dependent protein kinase II (CaMKII), may be a mediator of TKI-induced effects.Methods: CF viability in response to chronic treatment with both drugs was assessed using MTT assays and flow cytometry analysis. Calcium mobilization was assessed in CFs loaded with Fluo4-AM and CaMKII activation via oxidation was measured via quantitative immunoblotting. Effects of both drugs on mitochondrial function was determined by live mitochondrial imaging using MitoSOX red.Results: Treatment of CFs with sunitinib (0.1–10 μM) resulted in concentration-dependent alterations in CF phenotype, with progressively significant cell loss at higher concentrations. Flow cytometry analysis and MTT assays revealed increased cell apoptosis and necrosis with increasing concentrations of sunitinib. In contrast, equivalent concentrations of imatinib resulted in no significant change in cell viability. Both sunitinib and imatinib pre-treatment increased Angiotensin II-induced intracellular Ca2+ mobilization, with only sunitinib resulting in a significant effect and also causing increased CaMKII activation via oxidation. Live cell mitochondrial imaging using MitoSOX red revealed that both sunitinib and imatinib increased mitochondrial superoxide production in a concentration-dependent manner. This effect in response to both drugs was suppressed in the presence of the CaMKII inhibitor KN-93.Conclusions: Sunitinib and imatinib showed differential effects on CFs, with sunitinib causing marked changes in cell viability at concentrations where imatinib had no effect. Sunitinib caused a significant increase in Angiotensin II-induced intracellular Ca2+ mobilization and both TKIs caused increased mitochondrial superoxide production. Targeted CaMKII inhibition reversed the TKI-induced mitochondrial damage. These findings highlight a new role for CaMKII in TKI-induced cardiotoxicity, particularly at the level of the mitochondria, and confirm differential off-target toxicity in CFs, consistent with the differential selectivity of sunitinib and imatinib.

Lnc-Ang362 is a pro-fibrotic long non-coding RNA promoting cardiac fibrosis after myocardial infarction by suppressing Smad7

BackgroundCardiac fibrosis following myocardial infarction (MI) leads to cardiac remodeling and dysfunction. Dysregulation of Smad7 which negatively regulates the profibrotic transforming growth factor-β1 (TGF-β1)/Smad signaling promotes cardiac fibrosis. However, the molecular mechanisms underlying TGF-β1/Smad7 dysregulation remain elusive. Long non-coding RNAs (lncRNAs) are recently emerging as important regulators of cardiac diseases. Here, we report lnc-Ang362 is a novel lncRNA mediating MI-induced fibrosis through TGF-β1/Smad7 signaling pathway. Methods and resultsThe MI model was established by artificial coronary artery occlusion in rats. Microarray analysis identified 215 lncRNAs (fold change > 2.0, P < 0.05) differentially expressed between MI hearts and the sham group 4 weeks after MI. Lnc-Ang362 had the highest fold upregulation and the change was validated by reverse transcription polymerase chain reaction. Also, MI caused a marked increase in TGF-β1 and collagen I/III expression, but significantly downregulated Smad7 expression. Adult rat cardiac fibroblasts (RCFs) treated with TGF-β1 showed increased lnc-Ang362 expression and decreased Smad7 expression. Moreover, overexpression and knockdown of lnc-Ang362 by small interfering RNAs reduced and increased Smad7 expression, respectively. Importantly, this result was negatively correlated with the expression of collagen I/III in RCFs. Furthermore, the luciferase reporter assays confirmed that Smad7 was a validated lnc-Ang362 target. Further silencing Smad7 attenuated the effects of lnc-Ang362 knockdown on decreasing collagen I/III expression in RCFs. ConclusionsThese results suggested lnc-Ang362 promoted cardiac fibrosis after MI via directly suppressing Smad7, which may decrease the inhibitory feedback regulation of TGF-β1/Smad signaling pathway. Thus, lnc-Ang362 may be a novel profibrotic lncRNA in the regulation of cardiac fibrosis post MI.

Secreted Phospholipase A2-IIA Modulates Transdifferentiation of Cardiac Fibroblast through EGFR Transactivation: An Inflammation–Fibrosis Link

Secreted phospholipase A2-IIA (sPLA2-IIA) is a pro-inflammatory protein associated with cardiovascular disorders, whose functions and underlying mechanisms in cardiac remodelling are still under investigation. We herein study the role of sPLA2-IIA in cardiac fibroblast (CFs)-to-myofibroblast differentiation and fibrosis, two major features involved in cardiac remodelling, and also explore potential mechanisms involved. In a mice model of dilated cardiomyopathy (DCM) after autoimmune myocarditis, serum and cardiac sPLA2-IIA protein expression were found to be increased, together with elevated cardiac levels of the cross-linking enzyme lysyl oxidase (LOX) and reactive oxygen species (ROS) accumulation. Exogenous sPLA2-IIA treatment induced proliferation and differentiation of adult rat CFs. Molecular studies demonstrated that sPLA2-IIA promoted Src phosphorylation, shedding of the membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) ectodomain and EGFR phosphorylation, which triggered phosphorylation of ERK, P70S6K and rS6. This was also accompanied by an up-regulated expression of the bone morphogenic protein (BMP)-1, LOX and collagen I. ROS accumulation were also found to be increased in sPLA2-IIA-treated CFs. The presence of inhibitors of the Src/ADAMs-dependent HB-EGF shedding/EGFR pathway abolished the CF phenotype induced by sPLA2-IIA. In conclusion, sPLA2-IIA may promote myofibroblast differentiation through its ability to modulate EGFR transactivation and signalling as key mechanisms that underlie its biological and pro-fibrotic effects.

Regulation of connexin 43 by interleukin 1β in adult rat cardiac fibroblasts and effects in an adult rat cardiac myocyte: fibroblast co-culture model

Connexin 43 expression (Cx43) is increased in cardiac fibroblasts (CFs) following myocardial infarction. Here, potential mediators responsible for increasing Cx43 expression and effects of differential CF phenotype on cardiac myocyte (CM) function were investigated. Stimulating adult rat CFs with proinflammatory mediators revealed that interleukin 1β (IL-1β) significantly enhanced Cx43 levels through the IL-1β pathway. Additionally, IL-1β reduced mRNA levels of the myofibroblast (MF) markers: (i) connective tissue growth factor (CTGF) and (ii) α smooth muscle actin (αSMA), compared to control CFs. A co-culture adult rat CM:CF model was utilised to examine cell-to-cell interactions. Transfer of calcein from CMs to underlying CFs suggested functional gap junction formation. Functional analysis revealed contraction duration (CD) of CMs was shortened in co-culture with CFs, while treatment of CFs with IL-1β reduced this mechanical effect of co-culture. No effect on action potential rise time or duration of CMs cultured with control or IL-1β-treated CFs was observed. These data demonstrate that stimulating CFs with IL-1β increases Cx43 and reduces MF marker expression, suggesting altered cell phenotype. These changes may underlie the reduced mechanical effects of IL-1β treated CFs on CD of co-cultured CMs and therefore have an implication for our understanding of heterocellular interactions in cardiac disease.

β-Arrestin 2 mediates arginine vasopressin-induced IL-6 induction via the ERK1/2-NF-κB signal pathway in murine hearts.

Evidence to date suggests that β-arrestins act beyond their role as adapter proteins. Arginine vasopressin (AVP) may be a factor in inflammation and fibrosis in the pathogenesis of heart failure. In the present study we investigated the effect of AVP on inflammatory cytokine IL-6 production in murine hearts and the impact of β-arrestin 2-dependent signaling on AVP-induced IL-6 production. We found that administration of AVP (0.5 U/kg, iv) markedly increased the levels of IL-6 mRNA in rat hearts with the maximum level occurred at 6 h. In β-arrestin 2 KO mouse hearts, deletion of β-arrestin 2 decreased AVP-induced IL-6 mRNA expression. We then performed in vitro experiments in adult rat cardiac fibroblasts (ARCFs). We found that AVP (10−9–10−6 M) dose-dependently increased the expression of IL-6 mRNA and protein, activation of NF-κB signaling and ERK1/2 phosphorylation, whereas knockdown of β-arrestin 2 blocked AVP-induced IL-6 increase, NF-κB activation and ERK1/2 phosphorylation. Pharmacological blockade of ERK1/2 using PD98059 diminished AVP-induced NF-κB activation and IL-6 production. The selective V1A receptor antagonist SR49059 effectively blocked AVP-induced NF-κB phosphorylation and activation as well as IL-6 expression in ARCFs. In AVP-treated mice, pre-injection of SR49059 (2 mg/kg, iv) abolished AVP-induced NF-κB activation and IL-6 production in hearts. The above results suggest that AVP induces IL-6 induction in murine hearts via the V1A receptor-mediated β-arrestin2/ERK1/2/NF-κB pathway, thus reveal a novel mechanism of myocardial inflammation in heart failure involving the V1A/β-arrestin 2/ERK1/2/NF-κB signaling pathway.

Divergent Effects of Resveratrol on Rat Cardiac Fibroblasts and Cardiomyocytes.

In this study, we tested the potential cardioprotective effects of the phytoalexin resveratrol (Rsv) on primary adult rat cardiac fibroblasts (CF), myofibroblasts (MF) and cardiomyocytes. Adult rat CF and cardiomyocytes were isolated from male 10-week old Sprague–Dawley rats, cultured for either 24 h (cardiomyocytes) or 48 h (CF) before treatments. To isolate MF, CF were trypsinized after 48 h in culture, seeded in fresh plates and cultured for 24 h prior to treatment. All three cells were then treated for a further 24 h with a range of Rsv doses. In CF and MF, cell proliferation, viability, apoptosis assays were performed with or without Rsv treatment for 24 h. In cardiomyocytes, cell viability and apoptosis assay were performed 24 h after treatment. In separate experiments, CF was pre-incubated with estrogen, tamoxifen and fulvestrant for 30 min prior to Rsv treatment. Rsv treatment decreased proliferation of both fibroblasts and myofibroblasts. Rsv treatment also increased the proportion of dead CF and MF in a dose dependent manner. However, treatment with Rsv did not induce cell death in adult cardiomyocytes. There was an increase in the percentage of cells with condensed nuclei with Rsv treatment in both CF and MF, but not in cardiomyocytes. Treatment with estrogen, tamoxifen and fulvestrant alone or in combination with Rsv did not have any additional effects on CF survival. Our results demonstrate that treatment with Rsv can inhibit cell proliferation and induce cell death in rat CF and MF, while not affecting cardiomyocyte survival. We also demonstrated that the induction of cell death in CF with Rsv treatment was independent of estrogen receptor alpha (ERα) signaling.

Receptor tyrosine kinase inhibitors cause dysfunction in adult rat cardiac fibroblasts in vitro

The anti-cancer receptor tyrosine kinase inhibitors include known cardiotoxins: a component of this toxicity may be mediated by effects on cardiac fibroblasts (CFs). We hypothesised that imatinib mesylate (imatinib) and sunitinib malate (sunitinib) cause significant dysfunction in adult CFs. Following in vitro treatments with imatinib or sunitinib, adult rat CF viability was assessed by fluorescein diacetate assay, proliferation measured by bromodeoxyuridine nuclear incorporation and changes to the expression of CF secretome components determined by real time quantitative RT-PCR. Imatinib and sunitinib significantly reduced cell viability over 48 h, with EC50 values of 11.0 μM (imatinib) and 4.5 μM (sunitinib) respectively. Imatinib reduced CF proliferation from 35.5 ± 3.2% in control to 23.0 ± 5.5% (3 μM; p < 0.001) and to 9.4 ± 2.5% (10 μM; p < 0.001), whereas sunitinib reduced proliferation to 22.9 ± 3.1% (1 μM; p < 0.001) and to 15 ± 1.0% (3 μM; p < 0.001). Further, 10 μM imatinib increased mRNA expression of TGFB1 7-fold, (p < 0.01), IL6 6-fold (p < 0.01), and IL1B 7-fold (p < 0.05) and reduced PDGFD 15-fold (p < 0.01); whereas sunitinib specifically reduced IL1B mRNA expression 17-fold (p < 0.01). Overall, these findings show tyrosine kinase inhibitors cause significant dysfunction in CFs. These data point to an important role for the PDGF pathway in governing CF functions, including survival and proliferation.

Calcium Signal Pathway is Involved in Prostaglandin E2 Induced Cardiac Fibrosis in Cardiac Fibroblasts.

The present study was designed to explore the effects of PGE2 on cardiac fibrosis and the involved mechanism. We used western blot analysis, real-time quantitative PCR and immunostaining etc. to testify the mechanism. Our data showed that in cultured adult rat cardiac fibroblasts (CFs), PGE2 effectively promoted the expression of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF)，fibronectin (FN), Collagen I and induced [Ca2+]i increase. Besides, calcium increase evoked by PGE2 is mediated by virtue of EP1 activation. Instead of EP3 or EP4, inhibition of EP1 attenuated PGE2-stimulated upregulation of α-SMA，CTGF, FN, collagen I and [Ca2+]i, as well as the nuclear factor of activated T cell cytoplasmic 4 protein (NFATc4) translocation. PGE2 may promote cardiac fibrosis via EP1receptor and calcium signal pathway.

Ghrelin Ameliorates Angiotensin II-Induced Myocardial Fibrosis by Upregulating Peroxisome Proliferator-Activated Receptor Gamma in Young Male Rats

Angiotensin (Ang) II contributes to the formation and development of myocardial fibrosis. Ghrelin, a gut peptide, has demonstrated beneficial effects against cardiovascular disease. In the present study, we explored the effect and related mechanism of Ghrelin on myocardial fibrosis in Ang II-infused rats. Adult Sprague-Dawley (SD) rats were divided into 6 groups: Control, Ang II (200ng/kg/min, microinfusion), Ang II+Ghrelin (100 μg/kg, subcutaneously twice daily), Ang II+Ghrelin+GW9662 (a specific PPAR-γ inhibitor, 1 mg/kg/d, orally), Ang II+GW9662, and Ghrelin for 4 wks. In vitro, adult rat cardiac fibroblasts (CFs) were pretreated with or without Ghrelin, Ghrelin+GW9662, or anti-Transforming growth factor (TGF)-β1 antibody and then stimulated with or without Ang II (100 nmol/L) for 24 h. Ang II infusion significantly increased myocardial fibrosis, expression of collagen I, collagen III, and TGF-β1, as well as TGF-β1 downstream proteins p-Smad2, p-Smad3, TRAF6, and p-TAK1 (all p<0.05). Ghrelin attenuated these effects. Similar results were seen in Ang II-stimulated rat cardiac fibroblasts in vitro. In addition, Ghrelin upregulated PPAR-γ expression in vivo and in vitro, and treatment with GW9662 counteracted the effects of Ghrelin. In conclusion, Ghrelin ameliorated Ang II-induced myocardial fibrosis by upregulating PPAR-γ and in turn inhibiting TGF-β1signaling.

Abstract 470: Gata4, Mef2c and Tbx5 More Efficiently Transdifferentiate Endothelial Cells into Cardiomyocyte-like Cells Through Endothelial-Mesenchymal Transition Process

Background: The cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) have been used to successfully reprogram cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs). However their effects on other cell types remain largely unknown. Endothelial cells are an important population of the heart by Pinto et al. (2016). Given the more profound iCM induction effects observed in vivo, we have speculated that reprogramming of endogenous endothelial cells may also play an important role. Purpose: To investigate the cellular reprograming effects and mechanisms of GMT factors on cardiac endothelial cells and compare the outcomes with that from cardiac fibroblasts. Methods: Adult rat cardiac endothelial cells (REC) and cardiac fibroblasts (RCF) were purchased commercially. The cells were cultured in appropriate media and infected with GMT-expressing lentivirus and maintained with iCM media. Fourteen days later Immunofluorescence staining, FACS, and qRT-PCR were performed. Results: Both GMT treated RCFs and RECs showed positive staining of cardiac marker cTnT and αActinin-positive cells via immunofluorescence staining. FACS analysis showed GMT treated RECs had 16.7% of cTnT positive cells versus 3.3% in GMT treated RCF’s. qRT-PCR results demonstrated significant up-regulation of cTnT in GMT infected RECs (74.4±23.3 times, p&lt;0.05) as compared to the control group. Similar upregulation of cTnT was observed in RCFs but to a lesser degree (16.1±7.2 times, p=NS). Moreover, RECs presented a significant change in cell morphology from rounded cells in a cobblestone arrangement to elongated spindle-shaped cells during 14 days culture. In addition, qRT-PCR analysis demonstrated that endothelial-mesenchymal transition (EndoMT) markers such as Slug, Snail, Oct4, and αSMA were upregulated in the cells with GFP encoded virus infection and those markers were downregulated in the GMT-treated RECs (p&lt;0.01 for Snail, p&lt;0.05 for Slug, and αSMA). Conclusion: This report suggests that RECs are susceptive to GMT induced cardiac reprogramming than in RCFs. Our study also indicate that EMT and MET sequential process are involved in the reprogramming of RECs toward iCM cells. RECs could be targeted towards iCM generation for a higher reprogramming rate.

Abstract 349: Sall4 Blocks Cardiac Trans-differentiation but Stimulates Cardiac Stem-like Cell (iPSC) Generation and Improve Post MI Function In Vivo

Background: Sall4 is an essential component in maintaining embryonic stem cell self-renewal and pluripotency. Given its functional and protein level interaction with cardiac trans-differentiation factors Tbx5/Gata4, as well as its critical role in modulating embryonic cardiac development, we hypothesized that Sall4 may play an important role in cardiac reprogramming. Material/Method: We conducted gain- and loss- of function studies on neonatal and adult cardiac fibroblast from both mouse and rat. We also evaluated Sall4 function on infarcted rat heart. Results: Gata4/Mef2c/Tbx5 (GMT) administration markedly upregulated mRNA expression levels of stem factors including Sall4 , Bmi1 , and p63 , while inhibition of Sall4 largely enhanced GMT- mediated Cardiac troponin T ( cTNT ) induction. Conversely, overexpression of Sall4 plus Gata4/Mef2C in cardiac fibroblast for 7 days induces over 30-fold increase of Oct4 and Nanog by real-time PCR; longer culture induced rapidly-dividing iPSC-like clusters in both neonatal mouse and adult rat cardiac fibroblasts. Further tests showed that these iPSC-like cells were partially positive in alkaline phosphatases staining, capable of forming cardiosphere and differentiating into various cell types. Sall4 delivery also improved post-infarct cardiac function to a level that is comparable with Gata4 in rat. Conclusion: These results suggested that Sall4 is a promising target for cardiac reprogramming even though further characterization in vitro as well as in vivo is needed.

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.

Anti-cancer tyrosine kinase inhibitors increase oxidative stress in primary cardiac fibroblasts

Anti-cancer tyrosine kinase inhibitors (TKI) are known to exert cardiotoxicity that can result in heart failure. Here we have used primary adult rat cardiac fibroblasts (CFs) to examine whether imatinib and sunitinib (both TKIs) increase oxidative stress and cause increased oxidation and activation of calcium/calmodulin dependent protein kinase II (CaMKII). Activation of CaMKII is known to be important for cellular function but excessive activation is linked to cardiac pathology. CFs, isolated from adult Wistar rat hearts, were maintained in culture and treated over 24 hour with the TKIs sunitinib and imatinib (0.1–20 µM) in serum-free medium (DMEM). Cell phenotype and growth was monitored using a Nikon Eclipse (TE300) inverted microscope and ROS assays performed using a specially designed assay kit (Abcam, UK) which uses DCFDA to measure hydroxyl, peroxyl and other ROS activity. Quantitative immunoblotting was used to assess ox-CaMKII protein levels. Treatment with both TKIs caused a dose-dependent decrease in cell viability. Sunitinib exerted more toxic effects at lower concentrations than imatinib. Decreased cell viability was evident at concentrations as low as 1 µM for sunitinib with dramatic cell loss (>50%) evident at 10–20 µM. Significantly increased ROS levels (using the DFCDA-based assay) were observed in the presence of both imatinib (4956±102 vs 1984±358 (imatinib vs vehicle (a.u.), n=3, p In conclusion, both imatinib and sunitinib cause increased oxidative stress in adult primary CFs with resultant activation of CaMKII. These effects are evident at lower concentrations of sunitinib than for imatinib and reflect the increased cardiotoxicity of this TKI reported in the patient.

The Recombinant Human Secretory Leukocyte Protease Inhibitor (SLPI) protects cardiac fibroblasts injury against an in vitro ischemia/reperfusion injury

One of the major causes of cardiac cell death is an over-secretion of protease enzymes surrounding the ischemic tissue. Therefore, the inhibition of protease activity could be used as an alternative strategy to prevent the expansion of injury. In the present study, we investigated the pre-treatment effects of recombinant human Secretory Leukocyte Protease Inhibitor (rhSLPI) on simulated-ischemia/reperfusion (sI/R)-induced cardiac fibroblast cell death. The adult rat cardiac fibroblasts (ARCFs) were isolated from adult male Wistar rats (n=6). Isolated cells were characterized by through the detection of vimentin. Simulated ischemia/reperfusion was performed by incubating cells in an ischemic buffer for 40 min which were then reperfused with completed medium for 24 h. The cell viability was determined by MTT assay. The intracellular reactive oxygen species (ROS) production was measured by dichlorodihydrofluorescein assay. Activation of p38 MAPK was determined by Western blotting. The results showed that pre-treatment of rhSLPI at the concentrations of 400, 600, 800, and 1000 ng/mL significantly reduced sI/R-induced ARCFs death as well as intracellular ROS production. Moreover, pre-treatment of rhSLPI could reduce p38 MAPK activation and pro-apoptotic protein-Bax levels. In conclusion, pre-treatment of rhSLPI shows cardioprotective effects against sI/R-induced cardiac fibroblast cell death by reducing oxidative stress and reducing p38 MAPK activation.