Human Ventricular Fibroblasts Research Articles

Co-culture with normal and senescent AC16 cardiomyocytes modulates fibrotic response in primary human ventricular fibroblasts.

Transwell co-culture with human AC16 cardiomyocyte-like cells modifies the response of primary human ventricular fibroblasts to TGF-β stimulation. Fibrotic response markers including collagen I (COL1A1) and ɑ-smooth muscle actin (ACTA2) are amplified in the presence of AC16 cells, whereas others including periostin (POSTN) and fibronectin (FN1) are suppressed. Similar modulation is observed when the ventricular fibroblasts are co-cultured with AC16 cells under baseline and induced senescence conditions. Given that the response to TGF-β stimulation is commonly measured to study fibrotic signaling and drug treatments in vitro, the results here suggest that the effect of cellular crosstalk should be more broadly considered.

Atorvastatin attenuates TGF‑β1‑induced fibrogenesis by inhibiting Smad3 and MAPK signaling in human ventricular fibroblasts.

Excessive proliferation and myofibroblasts transformation of cardiac fibroblasts play a critical role in the process of cardiac fibrosis. Atorvastatin (ATV), a 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitor, is commonly used to treat hypercholesterolemia. It has previously been shown that ATV has potential anti-fibrotic effects. However, the underlying mechanisms of ATV against cardiac fibrosis remain to be fully elucidated, and to the best of our knowledge, there are no reports focusing on the effects of ATV on transforming growth factor-β1 (TGF-β1)-induced human ventricular fibroblasts (hVFs) activation. In the present study, hVFs were stimulated with TGF-β1 with or without pretreatment with ATV. Subsequently, hVF proliferation, cytotoxicity, myofibroblast differentiation and pro-fibrotic gene expression were assessed. Canonical and non-canonical signaling downstream of TGF-β1, such as Smad3 and mitogen-activated protein kinase (MAPK) signaling, were investigated by evaluating the phosphorylation levels of Smad3, extracellular signal-regulated kinase 1/2, p38 MAPK and c-Jun N-terminal kinase. The results indicated that ATV significantly prevented TGF-β1-induced cell proliferation, myofibroblast differentiation and production of extracellular matrix proteins, such as matrix metalloproteinase-2, collagen I and collagen III, in hVFs. Furthermore, ATV effectively inhibited TGF-β1-induced activation of Smad3 and MAPK signaling in hVFs. In conclusion, the present results demonstrated that ATV prevented TGF-β1-induced fibrogenesis in hVFs, at least in part by inhibiting the Smad3 and MAPK signaling pathways. Therefore, these results imply that ATV may be a promising agent to treat myocardial fibrosis.

Transient receptor potential channel TRPV4 mediates TGF-β1-induced differentiation of human ventricular fibroblasts.

Cardiac fibroblasts (CFs) are principal extracellular matrix-producing cells. In response to injury, CFs transdifferentiate into myofibroblasts. Intracellular calcium (Ca2+) signaling, involved in fibroblast proliferation and differentiation, is activated in fibroblasts through transient receptor potential (TRP) channels, but the function of these channels has not been investigated in human ventricular CFs. Under evaluation in this study, was the role of TRP channels in the differentiation of human ventricular CFs induced by transforming the growth factor beta (TGF-β), a pro-fibrotic cytokine. Human ventricular CFs were used in this study. The differentiation of CFs into myofibroblast was induced with TGF-β and was identified by the expression of smooth muscle actin. Results indicate that Ca2+ signaling was an essential component of ventricular CF dif-ferentiation. CFs treated with TGF-β demonstrated increased expression of a TRP channel, TRPV4, both at the mRNA and protein levels, which corresponded with CF-myofibroblast trans-differentiation, as evidenced by the upregulation of α-smooth muscle actin, a myofibroblast marker, and plasminogen activator inhibitor-1, which are fibrogenesis markers. An agonist of TRPV4 induced the conversion of CFs into myofibroblasts, whereas it's antagonist as well a Ca2+ chelating agent reduced it, indicating that the Ca2+ influx throughTRPV4 is required for CF trans-differentiation. Overall, these results dem-onstrate that TRPV4-mediated Ca2+ influx participates in regulating the differentiation of human ventricular CFs into myofibroblasts through the MAPK/ERK pathway. Overall, these results demonstrate that TRPV4-mediated Ca2+ influx participates in regulating the differentiation of human ventricular CFs into myofibroblasts through the MAPK/ERK pathway.

Comparative electrophysiology and pharmacology of hiPSC cardiomyocyte 2D mono-cultures and 2D co-cultures with human ventricular fibroblasts

Comparative electrophysiology and pharmacology of hiPSC cardiomyocyte 2D mono-cultures and 2D co-cultures with human ventricular fibroblasts

Abstract 119: Multi-Omics Investigation of Cardiomyocyte-to-Fibroblast Crosstalk in Human iPSC Models

Introduction: Cardiac cells communicate with each other in part through secreted proteins known as cardiokines. The total repertoire of proteins secreted by cardiac cells is unknown. We aim to apply human iPSC models and large-scale multi-omics to identify secreted proteins and the crosstalk signals they mediate. Method: We optimized an experimental protocol to recover and identify cell-specific secreted proteins from human iPSC-cardiac cells. Human iPSCs were differentiated into cardiomyocyte (CM) and endothelial cells (EC) using established protocols, after which secreted proteins were extracted from the conditioned medium for analysis. We combined multiplexed aptamer-based large-scale protein quantification platform and high-resolution mass spectrometry to identify secreted proteins from iPSC-CM, iPSC-ECs, and primary ventricular fibroblasts. An in-silico filter was implemented to prioritize bona fide cardiokines over proteins externalized by passive lysis. Result: We identified 146 candidate cardiokines at 1% false discovery rate, including cell-specific cardiokines as well as a common core secretome of three cardiac cells. We analyzed the data to identify potential signals secreted by cardiomyocytes to mediate fibroblast function, using a ligand-receptor model based on proteomics and single-cell transcriptomics data to prioritize cardiokines secreted by iPSC-CMs and which bind to fibroblast-expressed receptors. To examine their roles in fibrosis regulation, we selected three candidate cardiokines (PLAU, FGF7, and CXCL12) for verification using immunodetection, and exposed their recombinant proteins at multiple concentrations to human ventricular fibroblasts. The results nominated cardiokine-specific effects on recipient cell gene expression including evidence of modulation of fibroblast transcription and translation pathways. Conclusion: We demonstrate a multi-omics approach in iPSC models to explore the large-scale human secretome of multiple cardiac cell types. The approach holds promise for identifying disease markers and understanding intercellular communication in cardiac development and diseases.

Statins Reduce Cellular Respiration and Induce De‐differentiation of Myofibroblasts

BackgroundFibroblast to myofibroblast trans‐differentiation with altered bioenergetics precedes cardiac fibrosis (CF). Promotion of de‐differentiation could mitigate CF‐related pathologies; however, no specific remedial therapeutics for CF is clearly defined. Therefore, we determined whether statins, a hypolipidemic class of drugs commonly prescribed in patients at risk of heart failure (HF), can alter cellular bioenergetics and de‐differentiate myofibroblasts.MethodsPrimary cultures of differentiated human ventricular fibroblasts (hVFs) were randomly subjected to in vitro statin treatment (n = 3–6). Differentiation status was determined by α‐smooth muscle actin (α‐SMA) expression while cellular respiration was measured by Seahorse Extracellular Flux Analyzer XF‐96, as oxygen consumption rate (OCR) at baseline and following application of each well with mitochondrial modulators: Oligomycin (1 μg/ml), FCCP (0.1 μM) and antimycin A (1 μg/ml), normalized to total protein. Data were analyzed by either unpaired t test or one‐Way Analysis of Variance.ResultsIn vitro treatment of already differentiated myofibroblasts for 72 hours with both lipophilic (atorvastatin, simvastatin) and hydrophilic (rosuvastatin) statins concentration – dependently reduced α‐SMA/α‐β‐tubulin expression, normalized to % maximal differentiation. The respective IC50 values were 729 ± 13 nM (atorvastatin), 3.6 ± 1 μM (rosuvastatin) and 185 ± 13 nM (simvastatin); n = 3 per concentration of each statin. Basal OCR was significantly decreased in the simvastatin‐treated hVFs vs the differentiated control hVFs (TGF) (0.144 ± 0.026 vs. 0.278 ± 0.104, p = 0.002). Simvastatin reduced the ATP‐linked (0.083 ± 0.018 vs. 0.160 ± 0.059, p = 0.01), the proton leak‐linked (0.017 ± 0.008 vs. 0.074 ± 0.041, p = 0.01) and the maximal (0.250 ± 0.031 vs. 0.501 ± 0.142, p<0.001) OCR, but did not significantly affect spare capacity or non‐mitochondrial OCR. The inhibitory effect of simvastatin on the mitochondrial OCR was reversed by geranylgeranyl pyrophosphate (GGPP, 20 μM ) which significantly increased the basal (0.193 ± 0.053, p = 0.020), the spare‐capacity (0.233 ± 0.091, p = 0.008), and the maximal (0.377 ± 0.131, p = 0.003) OCR, without any change in the non‐mitochondrial OCR. The simvastatin treatment of the differentiated hVFs in vitro reduced ECAR after addition of FCCP (0.066 ± 0.011 vs. 0.093 ± 0.010, p<0.001) and AA (0.056 ± 0.014 vs. 0.097 ± 0.008, p<0.001). GGPP reversed ECAR to a similar level as control differentiated hVFs (FCCP: 0.081 ± 0.015, p = 0.005; AA: 0.080 ± 0.014, p = 0.018). Both atorvastatin (100 nM, 300 nM) and rosuvastatin (300 nM, 1 μM) also increased the ADP/ATP ratio in these cells, confirming the pan‐statin effect of decreased bioenergetics during de‐differentiation. Therefore, statins altered hVF bioenergetics and de‐differentiated the myofibroblasts which involved GGPP‐sensitive mechanisms, reduced cellular respiration with potential activation of KATP channels, as GGPP supplementation or KATP‐channel inhibition by glibenclamide countered the de‐differentiation.ConclusionStatin induced de‐differentiation of myofibroblasts via GGPP‐sensitive signaling, lowered bioenergetics, and KATP channels.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aging-related increase in store-operated Ca2+ influx in human ventricular fibroblasts.

Senescence-related fibrosis contributes to cardiac dysfunction. Profibrotic processes are Ca2+ dependent. The effect of aging on the Ca2+ mobilization processes of human ventricular fibroblasts (hVFs) is unclear. Therefore, we tested whether aging altered intracellular Ca2+ release and store-operated Ca2+ entry (SOCE). Disease-free hVFs from 2- to 63-yr-old trauma victims were assessed for cytosolic Ca2+ dynamics with fluo 3/confocal imaging. Angiotensin II or thapsigargin was used to release endoplasmic reticulum Ca2+ in Ca2+-free solution; CaCl2 (2 mM) was then added to assess SOCE, which was normalized to ionomycin-induced maximal Ca2+. The angiotensin II experiments were repeated after phosphoenolpyruvate pretreatment to determine the role of energy status. The expression of genes encoding SOCE-related ion channel subunits was assessed by quantitative PCR, and protein expression was assessed by immunoblot analysis. Age groups of <50 and ≥50 yr were compared using unpaired t-test or regression analysis. Ca2+ release by angiotensin II or thapsigargin was not different between the groups, but SOCE was significantly elevated in the ≥50-yr group. Regression analysis showed an age-dependent phosphoenolpyruvate-sensitive increase in SOCE of hVFs. Aging did not alter the mRNA expression of SOCE-related genes. The profibrotic phenotype of hVFs was evident by sprouty1 downregulation with age. Thus, an age-associated increase in angiotensin II- and thapsigargin-induced SOCE occurs in hVFs, independent of receptor mechanisms or alterations of mRNA expression level of SOCE-related ion channel subunits but related to the cellular bioenergetics status. Elucidation of mechanisms underlying enhanced hVF SOCE with aging may refine SOCE targets to limit aging-related progression of Ca2+-dependent cardiac fibrosis. NEW & NOTEWORTHY Human ventricular fibroblasts exhibit an age-related increase in store-operated Ca2+ influx induced by angiotensin II, an endogenous vasoactive hormone, or thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, independent of receptor mechanisms or genes encoding store-operated Ca2+ entry-related ion channel subunits. Selective inhibition of this augmented store-operated Ca2+ entry could therapeutically limit aging-related cardiac fibrosis.

Simvastatin reduces TGF-β1-induced SMAD2/3-dependent human ventricular fibroblasts differentiation: Role of protein phosphatase activation

BackgroundExcessive cardiac fibrosis due to maladaptive remodeling leads to progression of cardiac dysfunction and is modulated by TGF-β1-activated intracellular phospho-SMAD signaling effectors and transcription regulators. SMAD2/3 phosphorylation, regulated by protein-phosphatases, has been studied in different cell types, but its role in human ventricular fibroblasts (hVFs) is not defined as a target to reduce cytokine-mediated excessive fibrotic response and adverse cardiac remodeling. Statins are a class of drugs reported to reduce cardiac fibrosis, although underlying mechanisms are not completely understood. We aimed to assess whether simvastatin-mediated reduction in TGF-β1-augmented profibrotic response involves reduction in phospho-SMAD2/3 owing to activation of protein-phosphatase in hVFs. Methods and resultsCultures of hVFs were used. Effect of simvastatin on TGF-β1-treated hVF proliferation, cytotoxicity, myofibroblast differentiation/activation, profibrotic gene expression and protein-phosphatase activity was assessed. Simvastatin (1 μM) reduced effect of TGF-β1 (5 ng/mL) on hVF proliferation, myofibroblast differentiation (reduced α-smooth muscle actin [α-SMA-expression]) and activation (decreased procollagen-peptide release). Simvastatin also reduced TGF-β1-stimulated time-dependent increases in SMAD2/3 phosphorylation and nuclear translocation, mediated through catalytic activation of protein-phosphatases PPM1A and PP2A, which physically interact with SMAD2/3, thereby promoting their dephosphorylation. Effect of simvastatin on TGF-β1-induced fibroblast activation was annulled by okadaic acid, an inhibitor of protein-phosphatase. ConclusionsThis proof-of-concept study using an in vitro experimental cell culture model identifies the protective role of simvastatin against TGF-β1-induced hVF transformation into activated myofibroblasts through activation of protein phosphatase, a novel target that can be therapeutically modulated to curb excessive cardiac fibrosis associated with maladaptive cardiac remodeling.

248 - TRPV4 Has a Role as a Ca2+–Permeable Channel in the Differentiation of Human Ventricular Fibroblasts Into Myofibroblasts

Introduction: Cardiac fibroblasts (CFs) are the predominant cell types that reside in the interstitial space of the heart. In response to pathological stimuli, CFs are stimulated to undergo phenotypic modulation and become myofibroblasts that are hypersecretory and highly contractile and which deposit excessive extracellular matrix proteins. Ca2 + signals are essential to a diversity of cellular functions, including differentiation, gene expression, cell proliferation, and growth. CaV1.2-mediated L-type Ca2+ current (ICa,L) is a fundamental myocardial calcium entry channel, but CFs do not typically express the voltage-gated Ca2+ channel. Transient receptor potential (TRP) channels are nonselective calcium entry channels, and their function has been studied in many cell types. TRP is expressed in a cell-specific manner, and its role has not been evaluated in human ventricular fibroblasts. Methods: Here, we evaluate the role of the TRP channel in the differentiation of human ventricular fibroblasts into myofibroblasts. Results: Concordant with previous studies, Ca2+ was found in the current study to be an essential component in fibroblast differentiation. Intracellular chelation inhibited p-ERK expression and fibroblast differentiation. TRPV4 mRNA was expressed in human ventricular fibroblasts and increased on account of TGF-β1 (2 ng/mL). TRPV4 agonist (GSK1016790A) treatment induced the robust differentiation of fibroblasts into myofibroblasts. TRPV4 antagonist (RN-9893) significantly inhibited TGF-β1-induced p–ERK expression and the differentiation of CFs. Using ratio Ca2+ imaging measurements, we found a robust increase in Ca2+ influx after fibroblasts were treated with TRPV4 agonist (GSK1016790A). Conclusions: These results demonstrate that TRPV4 has a role as a Ca2+–permeable channel in the differentiation of human ventricular fibroblasts into myofibroblasts, through the ERK signaling pathway.

Abstract 110: Statin Therapy Alters the Transcriptome of Ventricular Fibroblasts From Human Failing Heart

Introduction: The mechanical and electrical dysfunction in heart failure (HF) is associated with excessive cardiac fibrosis (CF). Activation of human ventricular fibroblasts (hVF) and transdifferentiation to myofibroblasts underlies the increased CF. We recently reported that statin therapy reduced differentiation of hVF in HF patients. However, the underlying mechanism is not known. Therefore, we studied the effect of statin therapy on the transcriptome of hVF from HF patients. Hypothesis: We tested the hypothesis that statin therapy alters the expression of differentiation-associated transcription factors (TF) in hVFs from HF patients. Methods: Primary cultures of hVF obtained from HF patients undergoing left ventricular assist device implantation either under statin therapy for at least 1 year (n=3) or not (n=3). The extent of transcriptomic changes induced by statin therapy in hVFs was studied from total RNA using RT 2 Profiler TM PCR array - human transcription factors (Qiagen, Catalog No: PAHS-075Z ) run on Roche LightCycler 96-well block. Fold change was calculated by 2 -ΔΔCt method. Data were analyzed by Student’s t test, and P value &lt;0.05 was considered significant. Results: Out of the 84 related genes profiled, statin therapy upregulated significantly (P&lt;0.05) at least two-fold the following genes: CREB1 (Cyclic AMP-responsive element-binding protein 1), SMAD1, TCF7L2 (transcription factor 7-like 2 ), MEF2A(myocyte enhancer factor-2), ATF1(activating transcription factor 1), and SP3. CREB1, SMAD1, TCFL2, and MEF2A are mainly involved in signaling pathways of G-protein coupled receptors, bone morphogenetic proteins, Wnt, and mitogen-activated protein kinases/extracellular signal-regulated kinases, respectively, while ATF1 and SP3 are involved in various signaling pathways. TFAP2A (transcription factor AP-2 alpha) tends to be downregulated by two-fold, however, did not reach statistical significance. Conclusion: Statin therapy mitigates differentiation of hVFs from human failing heart patients by associated changes in the transcriptome. Selective targeting of hVF transcription factor may be a potential therapeutic strategy to de-differentiate myofibroblasts and mitigate the progression of CF and HF.

Neuregulin‐1β Alters the Context‐dependent Production of Immunomodulatory Factors in Human Ventricular Fibroblasts

Neuregulin‐1β (NRG‐1β) is a growth and survival factor critical for cardiac development and repair. We recently showed that NRG‐1β reduces cardiac fibrosis in infarcted swine and reduces basal and tumor necrosis factor β (TGFβ)‐induced α‐smooth muscle actin (α‐SMA) expression. In normal human ventricular fibroblasts, we also found that NRG‐1β caused a reduction in TGFβ‐induced α‐SMA, but we did not observe significantly lower α‐SMA levels in response to NRG‐1β under basal conditions. We thus hypothesized that the anti‐fibrotic effects of NRG‐1β might be context dependent. Indeed, when primary rat cardiac fibroblasts were cultured on softer substrates NRG‐1β had little effect on α‐SMA expression. NRG‐1β did not alter fibroblast collagen gel contraction in the presence or absence of TGFβ, nor did NRG‐1β treatment alter proliferation or viability of rodent or human fibroblasts. Nonetheless, these cells expressed all three NRG‐1β receptors (ErbB2, 3, and 4), and NRG‐1β alone reduced transcript levels of tumor necrosis factor‐α (TNF‐α) and prominin 1 (CD133) after 2 hours of treatment in the absence of serum. NRG‐1β inhibition of CXCL10, HLA‐DQB1, and adiponectin (ADIPOQ) on the other hand required the presence of TGFβ, as did NRG‐1β‐mediated increase of the TGFβ‐signaling inhibitor SMAD7. Most interesting was the induction of IL10 transcription in response to the combination of NRG‐1β and TGFβ, neither of which had any effect on IL10 transcript levels in the absence of the other. These results suggest that NRG‐1β modulates inflammation via cardiac fibroblast signaling and moreover that these signaling events are context‐dependent.Work was supported by the NIH (K01 HL121045; U01 HL100398).