Human Ventricular Cardiac Fibroblasts Research Articles

Chronic activation of human cardiac fibroblasts in vitro attenuates the reversibility of the myofibroblast phenotype

Activation of cardiac fibroblasts and differentiation to myofibroblasts underlies development of pathological cardiac fibrosis, leading to arrhythmias and heart failure. Myofibroblasts are characterised by increased α-smooth muscle actin (α-SMA) fibre expression, secretion of collagens and changes in proliferation. Transforming growth factor-beta (TGF-β) and increased mechanical stress can initiate myofibroblast activation. Reversibility of the myofibroblast phenotype has been observed in murine cells but has not been explored in human cardiac fibroblasts. In this study, chronically activated adult primary human ventricular cardiac fibroblasts and human induced pluripotent stem cell derived cFbs (hiPSC-cFbs) were used to investigate the potential for reversal of the myofibroblast phenotype using either subculture on soft substrates or TGF-β receptor inhibition. Culture on softer plates (25 or 2 kPa Young’s modulus) did not alter proliferation or reduce expression of α-SMA and collagen 1. Similarly, culture of myofibroblasts in the presence of TGF-β inhibitor did not reverse myofibroblasts back to a quiescent phenotype. Chronically activated hiPSC-cFbs also showed attenuated response to TGF-β receptor inhibition and inability to reverse to quiescent fibroblast phenotype. Our data demonstrate substantial loss of TGF-β signalling plasticity as well as a loss of feedback from the surrounding mechanical environment in chronically activated human myofibroblasts.

Biomimetic Substrate to Probe Dynamic Interplay of Topography and Stiffness on Cardiac Fibroblast Activation.

Materials with the ability to change properties can expand the capabilities of in vitro models of biological processes and diseases as it has become increasingly clear that static, stiff materials with smooth surfaces fall short in recapitulating the in vivo cellular microenvironment. Here, we introduce a patterned material that can be rapidly stiffened and softened in situ in response to an external magnetic field through the addition of magnetic inclusions into a soft silicone elastomer with topographic surface patterning. This substrate can be used for cell culture to investigate short-term cellular responses to dynamic stiffening or softening and the interaction with topography that encourages cells to assume a specific morphology. We investigated short-term cellular responses to dynamic stiffening or softening in human ventricular cardiac fibroblasts. Our results indicate that the combination of dynamic changes in stiffness with and without topographic cues induces different effects on the alignment and activation or deactivation of myofibroblasts. Cells cultured on patterned substrates exhibited a more aligned morphology than cells cultured on flat material; moreover, cell alignment was not dependent on substrate stiffness. On a patterned substrate, there was no significant change in the number of activated myofibroblasts when the material was temporally stiffened, but temporal softening caused a significant decrease in myofibroblast activation (50% to 38%), indicating a competing interaction of these characteristics on cell behavior. This material provides a unique in vitro platform to observe the time-dependent dynamics of cells by better mimicking more complex behaviors and realistic microenvironments for investigating biological processes, such as the development of fibrosis.

Abstract P1008: Bioelectric Sutures Generated From HiPSC-derived Cardiomyocytes Enable In Vitro Electrical Coupling Between Engineered Cardiac Tissues

Heart attack survivors live with permanent damage in their hearts and are at higher risk of developing heart failure. Current treatments do not directly repair or restore function in the damaged tissue. Implanting engineered cardiac tissues (ECTs) to restore contractility to the damaged myocardium is a promising therapy. However, this approach is challenged by poor integration between the implanted ECT and the host heart. To address this issue, we developed a bioelectric suture capable of propagating electrical signals to improve coupling between the two interfaces. We differentiated ventricular cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) following a well-adopted small molecule modulation of the Wnt signaling pathway. Metabolic-based lactate purification was performed to increase CM purity to > 80%, evaluated by flow cytometry analysis of cardiac troponin-T. We constructed fibrin threads by coextruding fibrinogen and thrombin through a blending tip connector into a HEPES bath. Polymerized, stretched, and dried threads were coated in Matrigel, clamped to suture heads, and seeded with hiPSC-CMs mixed with 5% human ventricular cardiac fibroblasts at a density of 5x10 5 cells/cm for 72 hours. We cultured the conductive and contractile sutures for 2 weeks under 1 Hz electrical stimulation. Optical mapping was performed to characterize electrical properties of bioelectric sutures, including action potential duration (APD) and conduction velocity (CV). Bioelectric sutures were electromechanically active with APD 100 = 547 +/- 29 ms and CV = 25.1 +/- 5.9 mm/s. a-actinin and connexin-43 staining revealed that hiPSC-CMs preferentially aligned with the thread and formed significant gap junctions, suggesting electrical syncytium formation. Preliminary results and ongoing work showed that bioelectric sutures sutured to two separate ECTs electrically coupled within 3 days, enabling directed electrical propagation from one tissue to the other. Collectively, these results demonstrate that bioelectric sutures establish a CM bridge for electrical propagation, which may improve the integration of implanted ECTs with the host heart and enable the development of therapies to treat diseases of cardiac electrical conduction.

Abstract 10973: Tenascin-C Deficiency Rescues the Effect of Diabetes on Cardiac and Vascular Dysfunction in Mice

Introduction: Tenascin-C (TN-C) upregulation is linked to worse clinical outcome in diabetic patients. However, the role of TN-C in the development of diabetic cardiomyopathy is still unknown. Hypothesis: Whether TN-C plays a maladaptive role in diabetic cardiomyopathy. Methods: AJ (n=14) and TNC-KO (n=16) adult male mice were injected with streptozotocin (50mg/kg) to induce diabetes. Cardiac function was measured by echocardiography at 16-18 weeks follow-up. Additionally, cardiomyocyte passive stiffness , vascular endothelial function (aorta), and cardiac fibrosis were assessed. In addition, the hemodynamic effect of human recombinant TN-C (hrTN-C; 80 ng/ml) on the isolated working rat heart (n=3) was evaluated. Isolated primary mouse cardiomyocytes (IPMC) were used to assess TN-C expression. Human ventricular cardiac fibroblasts (HCF) were cultured and treated with 1) TGF-β (20ng/ml); 2) rhTN-C (10μg/ml) and TLR4 inhibitor (TAK-242, 50 mM) in combination with TN-C and mRNA expression of α-SMA, TN-C, Col-1, Col-3 and ACE1 were assessed by RT-qPCR. Finally, HUVEC were treated either with rhTN-C (10μg/ml) or combination with TLR-4 inhibitor and analysed of the expression of NADPH oxidase 1 and 4 (NOX1, NOX4). Results: TN-C deficiency was accompanied by preserved LV ejection fraction and endothelium function in aorta (p<0.05, respectively). Diabetic TN-C KO mice show reduction in cardiomyocyte stiffness and diastolic dysfunction as compared to AJ diabetic group (p<0.01). Histology revealed less cardiac and perivascular fibrosis in diabetic TN-C KO animals than in the AJ diabetic group (p<0.01). In addition, rhTN-C resulted in reduction in cardiac function in isolated rat hearts. mRNA expression of TN-C was increased in IPMC. Moreover, TGF-β treatment markedly upregulated TN-C expression (p<0.01) and rhTN-C promoted pro-fibrotic genes expression (p<0.05, respectively). HUVEC incubated with rhTN-C showed increased expression of IL-6 and NOX4 via TLR-4 signalling. Conclusions: TN-C promotes a hostile environment that facilitates fibrosis and oxidative stress leading to cardiomyocyte and endothelial cell dysfunction. Thus, TN-C may play a crucial role in cardiovascular dysfunction in diabetes as well as a potential target for therapy.

Yes-Associated Protein (Yap) Is Up-Regulated in Heart Failure and Promotes Cardiac Fibroblast Proliferation

Left ventricular (LV) heart failure (HF) is a significant and increasing cause of death worldwide. HF is characterized by myocardial remodeling and excessive fibrosis. Transcriptional co-activator Yes-associated protein (Yap), the downstream effector of HIPPO signaling pathway, is an essential factor in cardiomyocyte survival; however, its status in human LV HF is not entirely elucidated. Here, we report that Yap is elevated in LV tissue of patients with HF, and is associated with down-regulation of its upstream inhibitor HIPPO component large tumor suppressor 1 (LATS1) activation as well as upregulation of the fibrosis marker connective tissue growth factor (CTGF). Applying the established profibrotic combined stress of TGFβ and hypoxia to human ventricular cardiac fibroblasts in vitro increased Yap protein levels, down-regulated LATS1 activation, increased cell proliferation and collagen I production, and decreased ribosomal protein S6 and S6 kinase phosphorylation, a hallmark of mTOR activation, without any significant effect on mTOR and raptor protein expression or phosphorylation of mTOR or 4E-binding protein 1 (4EBP1), a downstream effector of mTOR pathway. As previously reported in various cell types, TGFβ/hypoxia also enhanced cardiac fibroblast Akt and ERK1/2 phosphorylation, which was similar to our observation in LV tissues from HF patients. Further, depletion of Yap reduced TGFβ/hypoxia-induced cardiac fibroblast proliferation and Akt phosphorylation at Ser 473 and Thr308, without any significant effect on TGFβ/hypoxia-induced ERK1/2 activation or reduction in S6 and S6 kinase activities. Taken together, these data demonstrate that Yap is a mediator that promotes human cardiac fibroblast proliferation and suggest its possible contribution to remodeling of the LV, opening the door to further studies to decipher the cell-specific roles of Yap signaling in human HF.

Microphysiological System for High-Throughput Computer Vision Measurement of Microtissue Contraction.

The ability to measure microtissue contraction in vitro can provide important information when modeling cardiac, cardiovascular, respiratory, digestive, dermal, and skeletal tissues. However, measuring tissue contraction in vitro often requires the use of high number of cells per tissue construct along with time-consuming microscopy and image analysis. Here, we present an inexpensive, versatile, high-throughput platform to measure microtissue contraction in a 96-well plate configuration using one-step batch imaging. More specifically, optical fiber microprobes are embedded in microtissues, and contraction is measured as a function of the deflection of optical signals emitted from the end of the fibers. Signals can be measured from all the filled wells on the plate simultaneously using a digital camera. An algorithm uses pixel-based image analysis and computer vision techniques for the accurate multiwell quantification of positional changes in the optical microprobes caused by the contraction of the microtissues. Microtissue constructs containing 20,000-100,000 human ventricular cardiac fibroblasts (NHCF-V) in 6 mg/mL collagen type I showed contractile displacements ranging from 20-200 μm. This highly sensitive and versatile platform can be used for the high-throughput screening of microtissues in disease modeling, drug screening for therapeutics, physiology research, and safety pharmacology.

A new player in the game: treatment with antagomiR-21a-5p significantly attenuates histological and echocardiographic effects of experimental autoimmune myocarditis.

Myocarditis is associated with formidable symptoms and increased risk of adverse outcomes. Current approaches mostly rely on symptomatic treatments, warranting novel concepts for clinical practice. The aim of this study was to investigate the microRNA (miRNA) expression profile of Balb/c mice with experimental autoimmune myocarditis (EAM), choose a representative miRNA to antagonize after review of available literature and test its effects on myocardial inflammation in vitro and in vivo. Phase 1: EAM was induced in 12 male Balb/c mice, 10 animals served as controls. After sacrifice, next-generation sequencing (NGS) of the miRNA expression profile was performed. Based on these results, H9C2 cells and human ventricular cardiac fibroblasts exposed to lipopolysaccharide (LPS) were treated with the selected candidate antagomiR-21a-5p. Phase 2: EAM was induced in 48 animals. Thereof, 24 animals were either treated with antagomiR-21a-5p or negative control oligonucleotide in a nanoparticle formulation. Transthoracic echocardiography (TTE) was performed on Days 0, 7, 14, and 21. Histopathological examination was performed after sacrifice. Phase 1: EAM resulted in a significant up-regulation of 27 miRNAs, including miR-21a-5p (log2FC: 2.23, adj. P = 0.0026). Transfection with antagomiR-21a-5p resulted in a significant reduction of TNFα, IL-6, and collagen I in vitro. Phase 2: Treatment with antagomiR-21a-5p, formulated in polymeric nanoparticles for systemic injection, significantly attenuated myocardial inflammation (P = 0.001) and fibrosis (P = 0.013), as well as myocardial 'hypertrophy' on TTE. Silencing of miR-21a-5p results in a significant reduction of the expression of pro-inflammatory cytokines in vitro, as well as a significant attenuation of inflammation, fibrosis and echocardiographic effects of EAM in vivo.

Two in one: Neuregulin 1 improves cardiac diastolic and kindney funtcion in chronic kidney disease in rats

Abstract Introduction The prevalence of chronic renal disease (CKD) is continuously increasing in developed countries. Uremic cardiomyopathy characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction (DD) is a common cardiovascular complication of CKD. Cardiac microvascular low-grade inflammation and altered expression of endothelium derived Neuregulin-1 (NRG-1) are contributed to left ventricular DD. Our aim was to charachterize the effects of CKD on the expression of NRG-1 and 2) NRG-1 treatment on myocardial hypertrophy, diastolic dysfunction and renal function in the rat model of CKD. Methods Male Wistar rats were used and randomized into 3 groups: 1) Sham-operated,2) CKD induced by 5/6 nephrectomy (CKD) and 3) NRG-1-treated CKD group (CKD+NRG-1). In this group, 2 weeks after the CKD induction, the rats were treated with recombinant human NRG-1 (rhNRG-1) at the dose of 10 μg/kg/d for consecutive 10 days with tail vein injection of NRG-1. Serum and urea creatinine levels were measured to verify the development of CKD and transthoracic echocardiography was performed to monitor cardiac morphology and function. Furthermore, total RNA was isolated and RT-qPCR was performed to evaluate the expression levels of inflammatory chemokine and cytokines (TNF-α, TGF-β). In addition, NRG-1 protein levels were assessed in both kidney and heart tissue by ELISA. To clarify the underling anti-fibrotic mechanism, human ventricular cardiac fibroblasts (HCF) were cultured and treated with the TGF-β (20 ng/ml), and TGF-β + hrNRG-1 for 24 h, respectively. Confocal microscopy was used to detect α-smooth muscle actin (α-SMA) expression, marker for fibroblast to myofibroblast transtion. Results 10 weeks after the 5/6 nephrectomy, serum carbamide and creatinine levels were significantly increased and creatinine clearence was significantly decreased as compared to sham-operated animals proving the development of chronic kidney disease (CKD). This was accompanied by a significant decrease in NRG-1 protein expression levels in both cardiac and kidney tissue. Of note, NRG-1 treatment markedly reduced these changes, suggesting its renoprotective effects in CKD. In addition, In CKD animals, the significantly increased anterior, posterior and septal wall thicknesses with decreased end-diastolic and end-systolic diameters proved the development of concentric left ventricular hypertrophy. In CKD, the septal e' was significantly decreased and E/e' increased indicating the developemnt of diastolic dysfunction. These parameters were significantly improved by NRG-1 treatment. Mechanistically, NRG-1 treatment reduced the expression of inflammatory cytokines in compared to untreat group. Furthermore, TGF-β induced α-SMA and Col I upregulation was markedly reduced by hrNRG-1 treatment. Conclusions NRG-1 treatment improved both renal and cardiac funtion in CKD, via a mechansim including the anti-inflammatory and anti-fibrotic properties of NRG-1. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Österreichischer Austauschdienst

Rhein, a novel Histone Deacetylase (HDAC) inhibitor with antifibrotic potency in human myocardial fibrosis

Although fibrosis depicts a reparative mechanism, maladaptation of the heart due to excessive production of extracellular matrix accelerates cardiac dysfunction. The anthraquinone Rhein was examined for its anti-fibrotic potency to mitigate cardiac fibroblast-to-myofibroblast transition (FMT). Primary human ventricular cardiac fibroblasts were subjected to hypoxia and characterized with proteomics, transcriptomics and cell functional techniques. Knowledge based analyses of the omics data revealed a modulation of fibrosis-associated pathways and cell cycle due to Rhein administration during hypoxia, whereas p53 and p21 were identified as upstream regulators involved in the manifestation of cardiac fibroblast phenotypes. Mechanistically, Rhein acts inhibitory on HDAC classes I/II as enzymatic inhibitor. Rhein-mediated cellular effects were linked to the histone deacetylase (HDAC)-dependent protein stabilization of p53 under normoxic but not hypoxic conditions. Functionally, Rhein inhibited collagen contraction, indicating anti-fibrotic property in cardiac remodeling. This was accompanied by increased abundance of SMAD7, but not SMAD2/3, and consistently SMAD-specific E3 ubiquitin ligase SMURF2. In conclusion, this study identifies Rhein as a novel potent direct HDAC inhibitor that may contribute to the treatment of cardiac fibrosis as anti-fibrotic agent. As readily available drug with approved safety, Rhein constitutes a promising potential therapeutic approach in the supplemental and protective intervention of cardiac fibrosis.

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.

Heparin Augmentation Enhances Bioactive Properties of Acellular Extracellular Matrix Scaffold.

Extracellular matrix (ECM) maintains a reservoir of bioactive growth factors and matricellular proteins that provide bioinductive effects on local cells that influence phenotype and behaviors. Bioactive acellular ECM scaffolds can be used therapeutically to stimulate adaptive tissue repair. Fibroblast growth factor-2 (FGF-2) attenuates transforming growth factor-β1 (TGF-β1)-mediated cardiac fibrosis. Heparin glycosaminoglycan can influence FGF-2 bioactivity and could be leveraged to enhance tissue engineering strategies. We explored the effects of heparin on FGF-2 enhancement of bioactive ECM scaffold biomaterials for its antifibrotic effect on attenuating human cardiac myofibroblast activation. Increasing heparin concentration at a fixed concentration of FGF-2 markedly increased the amount of FGF-2 retained and eluted by ECM scaffolds. To explore synergistic bioinductive effects of heparin and FGF-2, collagen gel contraction assay using human cardiac myofibroblasts was performed in vitro. Myofibroblast activation was induced by profibrotic cytokine, TGF-β1. FGF-2 and heparin in combination reduced human cardiac myofibroblast-mediated collagen gel contraction to a greater extent than FGF-2 alone. These observations were confirmed for both human atrial and human ventricular cardiac fibroblasts. Cell death was not different between groups. In summary, heparin is an effective adjuvant to enhance FGF-2 loading and elution of acellular ECM scaffold biomaterials. Heparin increases the bioactive effects of FGF-2 in attenuating human cardiac myofibroblast activation in response to profibrotic TGF-β1. These data may inform tissue engineering strategies for myocardial repair to prevent fibrosis.

Egr-1 Upregulates Siva-1 Expression and Induces Cardiac Fibroblast Apoptosis

The early growth response transcription factor Egr-1 controls cell specific responses to proliferation, differentiation and apoptosis. Expression of Egr-1 and downstream transcription is closely controlled and cell specific upregulation induced by processes such as hypoxia and ischemia has been previously linked to multiple aspects of cardiovascular injury. In this study, we showed constitutive expression of Egr-1 in cultured human ventricular cardiac fibroblasts, used adenoviral mediated gene transfer to study the effects of continuous Egr-1 overexpression and studied downstream transcription by Western blotting, immunohistochemistry and siRNA transfection. Apoptosis was assessed by fluorescence microscopy and flow cytometry in the presence of caspase inhibitors. Overexpression of Egr-1 directly induced apoptosis associated with caspase activation in human cardiac fibroblast cultures in vitro assessed by fluorescence microscopy and flow cytometry. Apoptotic induction was associated with a caspase activation associated loss of mitochondrial membrane potential and transient downstream transcriptional up-regulation of the pro-apoptotic gene product Siva-1. Suppression of Siva-1 induction by siRNA partially reversed Egr-1 mediated loss of cell viability. These findings suggest a previously unknown role for Egr-1 and transcriptional regulation of Siva-1 in the control of cardiac accessory cell death.