Adult Cardiac Fibroblasts Research Articles

Internally-crosslinked alginate dialdehyde/alginate/gelatin-based hydrogels as bioprinting inks for prospective cardiac tissue engineering applications

Cardiovascular diseases represent a worldwide social and economic challenge, due to heart tissue limited regenerative capabilities. 3D bioprinted cell-laden constructs are a promising approach to develop patches for cardiac regeneration or in vitro models for new drug preclinical discovery and validation. However, the development of bioinks with optimal mechanical, rheological, and biological properties is still a challenge. Although alginate (Alg)-based bioinks have been extensively explored, such hydrogels lack cell adhesion properties and degradability. Additionally, 3D Alg structures are usually obtained by micro-extrusion bioprinting exploiting conventional external cross-linking methods, which introduce inhomogeneities and unpredictability in construct formation. This work exploits Alg internal ionic gelation mechanism to obtain homogeneous self-standing multilayered 3D printed constructs without employing support baths or post-printing crosslinking treatments, combining an insoluble calcium salt (calcium carbonate, CaCO3) with an acidifying agent (D-(+)-Glucono-1,5-lactone, GDL) to promote controlled release of calcium ions. Alg was blended with oxidized alginate (ADA) and gelatin (Gel) to achieve degradable and cell adhesive hydrogels for cardiac tissue engineering. Firstly, ADA/Alg bioink composition was tailored by varying polymer weight ratio (keeping ADA as the main component) and calcium ions concentration to achieve cardiac tissue-like viscoelastic properties. Then, the amount of Gel in ADA/Alg hydrogels was optimized to support adult human cardiac fibroblasts (AHCFs) adhesion, producing shear thinning inks with tunable viscoelastic properties (G’ 650-1300 Pa) and degradation profile (40-80% weight loss after 21 days in phosphate buffered saline, PBS) by varying Gel concentration. ADA/Alg/Gel hydrogels showed a shear thinning behavior suitable for 3D bioprinting and dependent on ink stabilization time, due to the gradual pH-triggered release of calcium ions over time. AHCFs-laden ADA/Alg/Gel bioinks could be successfully printed producing scaffolds with high shape fidelity and good cell viability post-printing. Finally, 3D AHCF-laden and H9C2-laden ADA/Alg/Gel bioinks with the highest gelatin content (25% w/w) allowed cell adhesion after 24 hours of incubation, showing potential application for cardiac tissue modelling. This research presented a comprehensive framework for advancing the design of bioink formulations enabling the printing of cell-adhesive and self-supporting constructs.

Mechanism of multifunctional adaptor protein SHARPIN regulating myocardial fibrosis and how SNP mutation affect the prognosis of myocardial infarction

Myocardial fibrosis (MF) is characterized by the excessive deposition of extracellular matrix within the heart, often following a cardiovascular insult. SHARPIN, a protein implicated in fibrosis, has emerged as a potential therapeutic target. This study aimed to elucidate the molecular mechanisms of SHARPIN in MF and to investigate the influence of its single nucleotide polymorphism (SNP), rs117299156, on myocardial infarction (MI) patients. A mouse model of Angiotensin II (AngII)-induced MF was established in SHARPIN heterozygous (SHARPIN+/−) and wild-type mice. Adult mouse cardiac fibroblasts (CFs) were isolated and subjected to adenovirus-encapsulated SHARPIN short hairpin RNA (shRNA) infection. Transcriptomic analysis was performed on CFs from SHARPIN+/− and wild-type (WT) mice, complemented by single-cell sequencing data from human cardiac tissues. Additionally, the association between the rs117299156 mutation and cardiovascular events in MI patients was assessed. Our findings indicate that SHARPIN is predominantly expressed in CFs and is upregulated in fibrotic myocardium. Partial knockdown of SHARPIN in murine hearts mitigated AngII-induced cardiac dysfunction and MF. Furthermore, reduced SHARPIN expression in CFs attenuated TGF-β1-induced collagen synthesis, cell proliferation, and myofibroblast transformation. Notably, MI patients carrying the rs117299156_C allele exhibited a reduced incidence of stroke events compared to those without the mutation.

Abstract We145: δ-catenin mRNA m 6 A Methylation Promotes Cardiac Fibrosis following Acute Myocardial Infarction

Background: Cardiac fibrosis is pivotal in heart failure progression, where excessive extracellular matrix (ECM) secretion by activated fibroblasts leads to adverse remodeling and dysfunction. While Wnt/β-catenin signaling influences fibroblast activation and cardiac fibrosis post-MI, its precise regulation remains unclear. Emerging evidence suggests N6-methyladenosine (m 6 A) mRNA methylation's role in disease pathology, yet its specific contribution to post-MI cardiac fibrosis is not well understood. Thus, we hypothesized that “ MI-induced METTL3 (a Key m6A mRNA methyltransferase) activation stabilizes δ-catenin mRNA, facilitating cardiac fibrosis and adverse remodeling ”. Methods: The mice underwent sham/MI surgeries for 4 weeks, after which heart tissues were collected for biochemical and histological analysis following heart function measurements. Additionally, methyl-immunoprecipitation followed by RNA sequencing (MeRIP-sequencing) was performed on the heart tissue post-surgery, and the data were analyzed to identify fibrosis-associated targets. Results: Ischemic injury significant increase in m6A mRNA methylation in heart tissues. This increase in m 6 A RNA was also observed in adult cardiac fibroblasts (AMFs) following TGFb treatment. Interestingly, METTL3 inhibition (METTL3 siRNA) resulted in a significant reduction in TGFb-induced periostin and fibronectin gene expression, while METTL3 overexpression enhanced the expression of fibrotic genes in AMFs. To identify differentially regulated m6A target genes, MeRIP-seq was performed on RNA isolated from mice hearts post-MI. The sequencing data suggested hypermethylation of fibrosis-associated genes, including δ-catenin, post-MI. Notably, TGFb-induced increased δ-catenin mRNA methylation led to δ-catenin mRNA stabilization. In contrast, METTL3 inhibition using siMETTL3 in AMFs and in METTL3 KO mice significantly reduced fibroblast activation and cardiac fibrosis. Conclusion: Our data suggests that hypermethylation of δ-catenin mRNA plays a significant role in the progression of cardiac fibrosis following AMI. Therefore, regulating METTL3 could be a potential therapeutic target for attenuating cardiac fibrosis.

Distinct phenotypes induced by acute hypoxia and TGF-β1 in human adult cardiac fibroblasts

Myocardial infarction (MI) causes hypoxic injury to downstream myocardial tissue, which initiates a wound healing response that replaces injured myocardial tissue with a scar. Wound healing is a complex process that consists of multiple phases, in which many different stimuli induce cardiac fibroblasts to differentiate into myofibroblasts and deposit new matrix. While this process is necessary to replace necrotic tissue, excessive and unresolved fibrosis is common post-MI and correlated with heart failure. Therefore, defining how cardiac fibroblast phenotypes are distinctly regulated by stimuli that are prevalent in the post-MI microenvironment, such as hypoxia and transforming growth factor-beta (TGF-β), is essential for understanding and ultimately mitigating pathological fibrosis. In this study, we acutely treated primary human adult cardiac fibroblasts with TGF-β1 or hypoxia and then characterized their phenotype through immunofluorescence, quantitative RT-PCR, and proteomic analysis. We found that fibroblasts responded to low oxygen with increased localization of hypoxia inducible factor 1 (HIF-1) to the nuclei after 4 h, which was followed by increased gene expression of vascular endothelial growth factor A (VEGFA), a known target of HIF-1, by 24 h. Both TGF-β1 and hypoxia inhibited proliferation after 24 h. TGF-β1 treatment also upregulated various fibrotic pathways. In contrast, hypoxia caused a reduction in several protein synthesis pathways, including collagen biosynthesis. Collectively, these data suggest that TGF-β1, but not acute hypoxia, robustly induces the differentiation of human cardiac fibroblasts into myofibroblasts. Discerning the overlapping and distinctive outcomes of TGF-β1 and hypoxia treatment is important for elucidating their roles in fibrotic remodeling post-MI and provides insight into potential therapeutic targets.

C-Src Facilitates ER-to-Mitochondria Ca2+ Transport and Activates Cardiac Fibroblasts under Pulmonary Arterial Hypertension

Introduction: Similar to left ventricular (LV) fibrosis, growing evidence suggests a potential link between right ventricular (RV) fibrosis, poor function of the pressure-overloaded RV, and mortality in patients with pulmonary arterial hypertension (PAH). However, no therapies are currently available that specifically target RV fibrosis. Although the pathological roles of an oxidative stress-sensitive tyrosine kinase c-Src in LV hypertrophy and failure are well characterized, it remains unclear whether c-Src is involved in RV pathology under PAH. Using heterologous expression systems, we previously showed that mitofusin 2 (Mfn2), a key component of the tethering structures between the endoplasmic reticulum (ER) and mitochondria (Mito) is a novel c-Src substrate in mitochondria; and c-Src-dependent tyrosine phosphorylation (P-Tyr) of Mfn2 at the outer mitochondrial membrane (OMM) decreases the ER-Mito distance, possibly through conformational changes of Mfn2, which facilitates ER-to-Mito Ca2+ transfer and increases reactive oxygen species (ROS). Hypothesis: c-Src signaling alters the size of ER-Mito microdomains and enhances oxidative stress in the RV during PAH. Methods: Primary adult cardiac fibroblasts (CFs) from human ventricles and a preclinical rat PAH model induced by Su gen 5416 injection and 3-week h ypo x ia followed by 4-week normoxia (SuHx) were used for in vitro and in vivo assays, respectively. RV tissues from pulmonary hypertension (PH) patients and sex-/aged-matched controls were used for biochemical and cell biological assays. Results: c-Src activation occurred specifically in CFs in the RV, but not in RV myocytes in a rat SuHx PAH model and human PH-RVs as assessed by immunohistochemistry, which is notably different from the reports on LV hypertrophy and failure. P-Tyr of Mfn2 was detectable from human PH-RVs by Western Blots. RV-CFs from PAH rats exhibited increased c-Src activity, decreased ER-Mito distance, increased ROS, and proliferative signaling activation compared to RV-CFs from control rats as assessed by live cell imaging, immunocytochemistry, and transmission electron microscopy. All of these changes in rat CFs from PAH-RVs were recapitulated by overexpressing c-Src in human CFs in vitro. In addition, c-Src significantly increased the speed and amount of mitochondrial Ca2+ uptake without changing cytosolic Ca2+ dynamics in human CFs, suggesting a potential link between c-Src activation, the size of ER-Mito microdomains, and RV fibrosis under PAH. To further dissect the role of c-Src activity in CFs especially in the mitochondria, we specifically inhibited c-Src activity at the OMM, where Mfn2 is located, using an OMM-targeted dominant-negative c-Src (termed mt-c-Src-DN) that was generated by the addition of an OMM-targeting sequence (amino acid 1-33 from human TOM20) to the N-terminus of kinase-dead c-Src-K295R/Y527F. The introduction of mt-c-Src-DN increased the ER-Mito distance and reduced the speed of ER-to-Mito Ca2+ transport in human CFs. Conclusion: c-Src-dependent P-Tyr of Mfn2 alters the ER-Mito tethering structure, facilitates ER-to-Mito Ca2+ transport, and increases mitochondrial ROS generation, which promotes CF activation in PAH. Therefore, CF- and mitochondria-specific inhibition of c-Src may provide an additional therapeutic strategy to attenuate RV fibrosis and failure in response to PAH. NIH R01HL136757 (to J.O.-U.), R01HL160699 (to B.S.J.), P30GM1114750 (Targeted project to J.O.-U.), P30GM110759 (Targeted project to J.O.-U.), U54GM115677 (Targeted project to B.S.J.), and American Heart Association (AHA) 18CDA34110091 (to B.S.J.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In Vitro Assessment of Cardiac Fibroblast Activation at Physiologic Stiffness.

Cardiac fibroblasts (CF) are an essential cell type in cardiac physiology, playing diverse roles in maintaining structural integrity, extracellular matrix (ECM) synthesis, and tissue repair. Under normal conditions, these cells reside in the interstitium in a quiescent state poised to sense and respond to injury by synthesizing and secreting collagen, vimentin, hyaluronan, and other ECM components. In response to mechanical and chemical stimuli, these "resident" fibroblasts can undergo a transformation through a continuum of activation states into what is commonly known as a "myofibroblast," in a process critical for injury response. Despite progress in understanding the contribution of fibroblasts to cardiac health and disease, much remains unknown about the signaling mediating this activation, in part owing to technical challenges in evaluating CF function and activation status in vitro. Given their role in monitoring the ECM, CFs are acutely sensitive to stiffness and pressure. High basal activation of isolated CFs is common due to the super-physiologic stiffness of traditional cell culture substrates, making assays dependent on quiescent cells challenging. To overcome this problem, cell culture parameters must be tightly controlled, and the use of dishes coated with biocompatible reduced-stiffness substrates, such as 8-kPa polydimethylsiloxane (PDMS), has shown promise in reducing basal activation of fibroblasts. Here, we describe cell culture protocol for maintaining CF quiescence in vitro to enable a dynamic range for the assessment of activation status in response to fibrogenic stimuli using PDMS-coated coverslips. Our protocol provides a cost-effective tool to study fibroblast signaling and activity, allowing researchers to better understand the underlying mechanisms involved in cardiac fibrosis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 8-kPa polydimethylsiloxane (PDMS)/gelatin-coated coverslips for cardiac fibroblast cell culture Basic Protocol 2: Isolation of adult cardiac fibroblasts and plating onto PDMS coverslips Basic Protocol 3: Assessment of cardiac fibroblast activation by α smooth muscle actin (αSMA) immunocytochemistry.

Abstract 17416: Epigenetic Regulation of Fibroblast Fate Transition in Cardiac Wound Healing

Background: Fibroblast transitioning between physiological and pathological states can define cardiac wound healing ability of the heart in response to stress. Preventing fibroblast activation and differentiation into myofibroblast formation can attenuate cardiac fibrosis and adverse cardiac remodeling. Changes in chromatin structure and accessibility are one of the essential drivers of gene expression/repression involved in myocardial injury. One such driver of chromatin remodeling is the group of polycomb proteins that alter expression of genes involved in cardiac wound healing including B cell specific Moloney murine leukemia virus integration site 1 (Bmi1), a component of the polycomb repressive complex 1 (PRC1). Bmi1 have been linked to self-renewal, proliferation, quiescence, and lineage commitment in many organs however, how Bmi1 can mediate fibroblasts states and mediate wound healing after cardiac injury is not understood. Hypothesis: Bmi1 mediates myofibroblast activation and transition, scar maturation, stability, and fibrotic responses by regulating histone marks after injury. Methods and Results: To establish the role of Bmi1 in cardiac wound healing, we developed an inducible knock out mouse model for Bmi1, which showed decrease in cardiac structure and function with maladaptive remodeling following myocardial injury. Bmi1 knockout (KO) mice showed significant decrease in ejection fraction and fractional shortening measured by echocardiography after cardiac injury. Concurrently, this significant decrease in heart function was associated with increased fibrosis and DNA damage analyzed using immunohistochemistry. Additionally, we found that the adult cardiac fibroblasts, isolated from the Bmi1 KO mice have increased expression of fibrotic genes including TGFβ, Acta2, and collagen using RTPCR analysis. Using single cell sequencing RNA and ATAC sequencing and cellular staining assays, we found significant changes in fibrotic signaling pathways of TGFβ. We found SMAD3 chromatin accessibility was increased with the loss of Bmi1 with the decrease in H2AK119Ub. Conclusion: Bmi1 mediates wound healing ability by regulating cardiac fibroblast transition after cardiac injury.

Abstract P3159: Exosomes Derived From Podoplanin Positive Cells Induce Endo-Mt And Myofibroblast Transformation In Healthy Mouse Heart And After Ischemic Injury

Specific understanding of the differences in cell communications between physiologicaland pathological conditions harbor the understanding of mechanisms to recover theequilibrium in the injured tissue and restore the organ function. Neurohumoral factorsreleased after myocardial infarction (MI) induce the expression of platelet aggregation-inducing type I transmembrane glycoprotein named Podoplanin (PDPN) in mesenchymalstromal cells, which pivot their structural support function and actively participate in theremodeling of the ischemic tissue releasing an abundance of extracellular vesicles calledexosomes. Exosomes (exo) derived from activated PDPN+ cells conditioned media wereused in vivo for the treatment of healthy mouse hearts, and in vitro for the treatment ofmouse cardiac endothelial cells (mCECs), NHI-3T3 and adult cardiac fibroblast. Ourongoing experiments showed that in vivo, PDPN-exo could independently alter healthyheart physiology and structure; mice hearts treated with PDPN-exo developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increaseof the end diastolic and systolic volumes. The fibrotic area was characterized by ECMdeposition, increased lymphatic vessels and infiltrating CD45+ cells. In vitro, PDPN-exoreprogramed mCECs, NHI-3T3 and adult cardiac fibroblast, upregulating the expressionof fibrotic markers suggesting an Endothelial-Mesenchymal Transition (EndoMT) andmyofibroblast transformation. Proteomic analysis of PDPN-exo suggests these transitionsmay depend upon the delivery of NOTCH1 via exosomes and the cleavage of it throughγ-Secretase. Experiments of gain and loss of NOTCH1 function showed thatoverexpression of NOTCH1 intracellular domain (NICD1) in vitro was sufficient to derivea lineage switching in mCECs and fibroblasts on the contrary in vivo delivery and in vitrotreatment with exosomes derived from PDPN+ cells silenced for NOTCH1 or treatmentwith γ-Secretase inhibitor did not alter the heart structure and function or the endothelialcells and fibroblast lineage. Thus, NOTCH1 delivery via PDPN-exo after MI play a majorrole in scar remodeling and its biology can be pharmacologically modulated.

Abstract P2113: Zdhhc7 Uniquely Influences Cardiac Fibroblast Activity Amongst Golgi-localized S-acyltransferases

Cardiac fibroblasts (CF’s), the heart’s most critical regulators of the extra-cellular matrix, can undergo a continuum of activation states when stimulated that influence their proliferation, migration, collagen production, and cell-to-cell communication in response to injury. The signaling pathways that underlie this transformation though, are poorly understood. Previous studies have shown zDHHC3, an S-acyltransferase enzyme that mediates the reversible lipid modification, palmitoylation, to be important in cardiac remodeling in cardiomyocytes due to its ability to regulate intracellular signaling, though the role of zDHHC enzymes and palmitoylation in CF signaling are unknown. To investigate this, we deleted zDHHC3 or the closely related enzyme, zDHHC7, in cardiac fibroblasts to screen for changes in fibroblast activity and myocardial fibrosis. Loss of zDHHC7, but not zDHHC3, resulted in increased expression of the activated cardiac fibroblast marker α-smooth muscle actin in response to TGFb stimulation and a trend towards increased mRNA levels of fibrotic genes. More notably, zDHHC7 deletion significantly blunted migratory ability of adult cardiac fibroblasts in scratch assays. These data suggest zDHHC7 plays a critical role in CF signaling, and future experiments include analyzing the impact of zDHHC3/7 deletion alone or in combination on cardiac fibrosis and identifying key substrates and pathways being modified by these enzymes in CFs.

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.

EphrinB2 drives osteogenic fate of adult cardiac fibroblasts in a calcium influx dependent manner.

Cardiac calcification is a crucial but underrecognized pathological process, greatly increasing the risk of cardiovascular diseases. Little is known about how cardiac fibroblasts, as a central mediator, facilitate abnormal mineralization. Erythropoietin-producing hepatoma interactor B2 (EphrinB2), previously identified as an angiogenic regulator, is involved in fibroblast activation, while its role in the osteogenic differentiation of cardiac fibroblasts is unknown. Bioinformatics analysis was conducted to characterize the expression of the Ephrin family in human calcified aortic valves and calcific mouse hearts. The effects of EphrinB2 on cardiac fibroblasts to adopt osteogenic fate was determined by gain- and loss-of-function. EphrinB2 mRNA level was downregulated in calcified aortic valves and mouse hearts. Knockdown of EphrinB2 attenuated mineral deposits in adult cardiac fibroblasts, whereas overexpression of EphrinB2 promoted their osteogenic differentiation. RNA sequencing data implied that Ca2+-related S100/receptor for advanced glycation end products (RAGE) signaling may mediate EphrinB2-induced mineralization in cardiac fibroblasts. Moreover, L-type calcium channel blockers inhibited osteogenic differentiation of cardiac fibroblasts, implying a critical role in Ca2+ influx. In conclusion, our data illustrated an unrecognized role of EphrinB2, which functions as a novel osteogenic regulator in the heart through Ca2+ signaling and could be a potential therapeutic target in cardiovascular calcification.NEW & NOTEWORTHY In this study, we observed that adult cardiac fibroblasts but not neonatal cardiac fibroblasts exhibit the ability of osteogenic differentiation. EphrinB2 promoted osteogenic differentiation of cardiac fibroblasts through activating Ca2+-related S100/RAGE signaling. Inhibition of Ca2+ influx using L-type calcium channel blockers inhibited EphrinB2-mediated calcification of cardiac fibroblasts. Our data implied an unrecognized role of EphrinB2 in regulating cardiac calcification though Ca2+-related signaling, suggesting a potential therapeutic target of cardiovascular calcification.

Neonatal and adult cardiac fibroblasts exhibit inherent differences in cardiac regenerative capacity

Directly reprogramming fibroblasts into cardiomyocytes improves cardiac function in the infarcted heart. However, the low efficacy of this approach hinders clinical applications. Unlike the adult mammalian heart, the neonatal heart has an intrinsic regenerative capacity. Consequently, we hypothesized that birth imposes fundamental changes in cardiac fibroblasts which limit their regenerative capabilities. In support, we found that reprogramming efficacy invitro was markedly lower with fibroblasts derived from adult mice versus those derived from neonatal mice. Notably, fibroblasts derived from adult mice expressed significantly higher levels of pro-angiogenic genes. Moreover, under conditions that promote angiogenesis, only fibroblasts derived from adult mice differentiated into tube-like structures. Targeted knockdown screening studies suggested a possible role for the transcription factor Epas1. Epas1 expression was higher in fibroblasts derived from adult mice, and Epas1 knockdown improved reprogramming efficacy in cultured adult cardiac fibroblasts. Promoter activity assays indicated that Epas1 functions as both a transcription repressor and an activator, inhibiting cardiomyocyte genes while activating angiogenic genes. Finally, the addition of an Epas1 targeting siRNA to the reprogramming cocktail markedly improved reprogramming efficacy invivo with both the number of reprogramming events and cardiac function being markedly improved. Collectively, our results highlight differences between neonatal and adult cardiac fibroblasts and the dual transcriptional activities of Epas1 related to reprogramming efficacy.

Dual Blockade of TGF-β Receptor and Endothelin Receptor Synergistically Inhibits Angiotensin II-Induced Myofibroblast Differentiation: Role of AT1R/Gαq-Mediated TGF-β1 and ET-1 Signaling.

Angiotensin II (Ang II) upregulates transforming growth factor-beta1 (TGF-β1) and endothelin-1 (ET-1) in various types of cells, and all of them act as profibrotic mediators. However, the signal transduction of angiotensin II receptor (ATR) for upregulation of TGF-β1 and ET-1, and their effectors that play an essential role in myofibroblast differentiation, are not fully understood. Therefore, we investigated the ATR networking with TGF-β1 and ET-1 and identified the signal transduction of these mediators by measuring the mRNA expression of alpha-smooth muscle actin (α-SMA) and collagen I using qRT-PCR. Myofibroblast phenotypes were monitored by α-SMA and stress fiber formation with fluorescence microscopy. Our findings suggested that Ang II induced collagen I and α-SMA synthesis and stress fiber formation through the AT1R/Gαq axis in adult human cardiac fibroblasts (HCFs). Following AT1R stimulation, Gαq protein, not Gβγ subunit, was required for upregulation of TGF-β1 and ET-1. Moreover, dual inhibition of TGF-β and ET-1 signaling completely inhibited Ang II-induced myofibroblast differentiation. The AT1R/Gαq cascade transduced signals to TGF-β1, which in turn upregulated ET-1 via the Smad- and ERK1/2-dependent pathways. ET-1 consecutively bound to and activated endothelin receptor type A (ETAR), leading to increases in collagen I and α-SMA synthesis and stress fiber formation. Remarkably, dual blockade of TGF-β receptor and ETR exhibited the restorative effects to reverse the myofibroblast phenotype induced by Ang II. Collectively, TGF-β1 and ET-1 are major effectors of AT1R/Gαq cascade, and therefore, negative regulation of TGF-β and ET-1 signaling represents a targeted therapeutic strategy for the prevention and restoration of cardiac fibrosis.

Inhibition of Eicosanoid Degradation Mitigates Fibrosis of the Heart.

Organ fibrosis due to excessive production of extracellular matrix by resident fibroblasts is estimated to contribute to >45% of deaths in the Western world, including those due to cardiovascular diseases such as heart failure. Here, we screened for small molecule inhibitors with a common ability to suppress activation of fibroblasts across organ systems. High-content imaging of cultured cardiac, pulmonary, and renal fibroblasts was used to identify nontoxic compounds that blocked induction of markers of activation in response to the profibrotic stimulus, transforming growth factor-β1. SW033291, which inhibits the eicosanoid-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase, was chosen for follow-up studies with cultured adult rat ventricular fibroblasts and human cardiac fibroblasts (CF), and for evaluation in mouse models of cardiac fibrosis and diastolic dysfunction. Additional mechanistic studies were performed with CFs treated with exogenous eicosanoids. Nine compounds, including SW033291, shared a common ability to suppress transforming growth factor-β1-mediated activation of cardiac, pulmonary, and renal fibroblasts. SW033291 dose-dependently inhibited transforming growth factor-β1-induced expression of activation markers (eg, α-smooth muscle actin and periostin) in adult rat ventricular fibroblasts and normal human CFs, and reduced contractile capacity of the cells. Remarkably, the 15-hydroxyprostaglandin dehydrogenase inhibitor also reversed constitutive activation of fibroblasts obtained from explanted hearts from patients with heart failure. SW033291 blocked cardiac fibrosis induced by angiotensin II infusion and ameliorated diastolic dysfunction in an alternative model of systemic hypertension driven by combined uninephrectomy and deoxycorticosterone acetate administration. Mechanistically, SW033291-mediated stimulation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase signaling was required for the compound to block CF activation. Of the 12 exogenous eicosanoids that were tested, only 12(S)-hydroxyeicosatetraenoic acid, which signals through the G protein-coupled receptor, GPR31, recapitulated the suppressive effects of SW033291 on CF activation. Inhibition of degradation of eicosanoids, arachidonic acid-derived fatty acids that signal through G protein-coupled receptors, is a potential therapeutic strategy for suppression of pathological organ fibrosis. In the heart, we propose that 15-hydroxyprostaglandin dehydrogenase inhibition triggers CF-derived autocrine/paracrine signaling by eicosanoids, including 12(S)-hydroxyeicosatetraenoic acid, to stimulate extracellular signal-regulated kinase 1/2 and block conversion of fibroblasts into activated cells that secrete excessive amounts of extracellular matrix and contribute to heart failure pathogenesis.

Abstract 12710: Adipocyte-Derived Exosomal Lncrna Sbf2-as1 Promotes Diabetic Myocardial Fibrosis Through Regulating Hnrnpa1/nrf2 Pathway via Sumoylation

Introduction: The inhibition of Nrf2 pathway is regarded as the dominant process that correlates with the pathogenesis of diabetic cardiomyopathy (DCM). Recently, accumulating evidence reveals that lncRNAs play key roles in regulating Nrf2 pathway. However, the underlying mechanisms remain unclear. Here, we found that adipocyte lncRNA SBF2-AS1 (SBF2-AS1) enhanced the SUMOylation of hnRNPA1, which subsequently facilitated the package of SBF2-AS1 into exosomes under high glucose conditions. Hypothesis: We hypothesized that AdEXO-SBF2-AS1 promotes diabetic myocardial fibrosis through regulating the hnRNPA1/Nrf2 pathway via SUMOylation. Methods: We examined the effect of exosomes from diabetic (db/db) mice-derived adipocytes on ANG-II-induced cardiac fibrosis and function in non-diabetic mice. In the invitro study, HG (33mmol/L)-induced AdEXO were cultured with adult human cardiac fibroblasts (aHCFs). Differentially expressed lncRNAs in AdEXO were screened using lncRNA sequencing. Results: Intramyocardial injection of diabetic AdEXO in the non-diabetic heart exacerbated myocardial fibrosis. Whereas administration of an exosomes biogenesis inhibitor attenuated cardiac fibrosis in diabetic mice. We identified SBF2-AS1 is a common molecule significantly increased in diabetic AdEXO and HG-stimulated non-diabetic AdEXO. After four weeks of ANG II infusion, EXO-db/dbWT-injected mice displayed significant fibrosis in the heart. However, interestingly, mice receiving SBF2-AS1-deficient db/db-EXO showed a decrease in cardiac fibrosis. Similarly, AdEXO-SBF2-AS1 promoted aHCFs proliferation and transformation capabilities in vitro. Mechanistically, SBF2-AS1 induced UBC9 overexpression to catalyze the SUMOylation of hnRNPA1, which facilitated its packaging into exosomes. Subsequently, exosomal SBF2-AS1 was specifically transmitted into aHCFs and epigenetically downregulated Nrf2 expression by decreasing H3K9 acetylation level in the Nrf2 promoter. Conclusions: Our findings highlight a novel mechanism whereby the AdEXO-mediated SBF2-AS1/hnRNPA1/Nrf2 regulatory axis promotes diabetic myocardial fibrosis in a SUMOylation-dependent manner and implicate SBF2-AS1 as an attractive therapeutic target for DCM.

Exercise training attenuates angiotensin II-induced cardiac fibrosis by reducing POU2F1 expression

BackgroundExercise training protects against heart failure. However, the mechanism underlying the protective effect of exercise training on angiotensin II (Ang II)-induced cardiac fibrosis remains unclear. MethodsAn exercise model involving C57BL/6N mice and 6 weeks of treadmill training was used. Ang II (1.44 mg/kg/day) was administered to induce cardiac fibrosis. RNA sequencing and bioinformatic analysis were used to identify the key factors mediating the effects of exercise training on cardiac fibrosis. Primary adult mouse cardiac fibroblasts (CFs) were used in vitro. Adeno-associated virus serotype 9 was used to overexpress POU domain, class 2, transcription factor 1 (POU2F1) in vivo. ResultsExercise training attenuated Ang II-induced cardiac fibrosis and reversed 39 gene expression changes. The transcription factor regulating the largest number of these genes was POU2F1. Compared to controls, POU2F1 was shown to be significantly upregulated by Ang II, which is itself reduced by exercise training. In vivo, POU2F1 overexpression nullified the benefits of exercise training on cardiac fibrosis. In CFs, POU2F1 promoted cardiac fibrosis. CCAAT enhancer-binding protein β (C/EBPβ) was predicted to be the transcription factor of POU2F1 and verified using a dual-luciferase reporter assay. In vivo, exercise training activated AMP-activated protein kinase (AMPK) and alleviated the increase in C/EBPβ induced by Ang II. In CFs, AMPK agonist inhibited the increase in C/EBPβ and POU2F1 induced by Ang II, whereas AMPK inhibitor reversed this effect. ConclusionExercise training attenuates Ang II-induced cardiac fibrosis by reducing POU2F1. Exercise training inhibits POU2F1 by activating AMPK, which is followed by the downregulation of C/EBPβ, the transcription factor of POU2F1.

Empagliflozin rescues primary murine cardiac fibroblasts, but not cardiomyocytes, from hypoxia-reoxygenation injury through STAT3-mediated cascades

Abstract Introduction Empagliflozin (EMPA) is a selective sodium glucose co-transporter 2 inhibitor (SGLT2i) and an established drug against type 2 diabetes mellitus. Cardiovascular clinical outcome trials on EMPA have shown a reduced mortality rate related to major cardiovascular events, but the precise mechanism of action remains elusive. We have previously proved that chronic EMPA administration reduces infarct size in an in vivo murine model of ischemia / reperfusion injury, irrespective of the diabetic status. The cardioprotective effect of EMPA was attributed to the increase of Signal Transducer and Activator of Transcription 3 (STAT3) phosphorylation. Purpose In this study we aimed to decipher the cell specific effects of EMPA on primary adult ventricular murine cardiomyocytes (pAVMCs) and primary murine cardiac fibroblasts (pMCFs), subjected to hypoxia/reoxygenation (HYP/REO) and to 1) investigate the effect of EMPA on cell death, and 2) reveal the mechanism of cardioprotection. Methods PAVMCs and pMCFs were isolated from adult C57BL/6 murine hearts (n=4–6) and were incubated with EMPA (500nM–100nM) for 24 hours. Cells were then subjected to 3 hours of HYP (99% N2), followed by 1 hour of REO. STATTIC, a selective STAT3 inhibitor was used (500nM–1μM) to determine the contribution of STAT3 on the observed effect. MTT assay was performed at the end of REO to determine cell viability. To unravel the mechanism of cardioprotection, the experimental protocol was repeated, and the cells were either stained with dihydroethidium (DHE) dye to determine relative oxidative stress changes or were harvested for isolation of total protein content. We focused on STAT3 and Akt kinase as key mediators of the cardioprotective survivor activating factor enhancement (SAFE) and the reperfusion injury salvage kinase (RISK) pathways. We also investigated the expression of Cardiotrophin-1 (CT-1), as a potential upstream mediator of STAT3 phosphorylation. Results The evaluation of cell death revealed that pMCFs viability was significantly increased upon EMPA treatment. Added to this, STATTIC co-treatment with EMPA blunted the protective effect on pMCFs, which indicates that EMPA's protection is STAT3-mediated. Oxidative stress remained unaltered by the treatments. Investigation of the molecular pathways that are responsible for the increased viability of pMCFs revealed that EMPA treatment induces an increased phosphorylation of STAT3 and an increase in CT-1 expression, which are both reversed by STATTIC. Akt phosphorylation and expression remained unchanged by EMPA treatment. Notably, the effect of EMPA on pAVMCs' viability was negligible. Conclusion(s) EMPA does not protect pAVMCs, from HYP/REO injury. EMPA rescues the pMCFs in a STAT-3 dependent manner which indicates that EMPA's cardioprotective effect is cell specific and implicates the SAFE pathway. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Boehringer Ingelheim International GmbHGrant title: “Investigation of the distinct effects of Empagliflozin on different cell populations of the healthy myocardium”

Abstract P2050: Acetyl-coa Synthesis Is Required For Chromatin Remodeling In Myofibroblast Differentiation And Persistence

Heart failure (HF) features fibrotic remodeling by myofibroblasts, a differentiated fibroblast population responsible for wound repair. Discovering mechanisms of myofibroblast formation and function may yield novel targets to delay or reverse HF. Myofibroblasts metabolically reprogram to mediate transcriptional activation of fibrotic genes through chromatin remodeling. We previously reported that α-ketoglutarate production for histone demethylation is essential for cardiac fibrosis, but metabolic regulation of histone acetylation in fibrosis is unclear. ATP-citrate lyase (ACLY) and Acetyl-coenzyme A synthetase 2 (ACSS2) synthesize acetyl-CoA in the cytoplasm and have also been reported to supply substrate for histone acetyltransferases in the nucleus to modify regulatory histone residues. To investigate acetyl-CoA metabolism in myofibroblast formation, we stimulated adult mouse and human cardiac fibroblasts (CFs) with the pro-fibrotic agonist transforming growth factor β (TGFβ) and treated cells with pharmacological inhibitors of ACLY and ACSS2. Either ACLY or ACSS2 inhibition sufficiently reverted differentiated myofibroblasts to a non-fibrotic phenotype, even during continuous TGFβ stimulation. Genetic deletion of ACLY in fibroblasts recapitulated these results. Further, ACLY inactivation was sufficient to decrease global histone acetylation. ACLY deletion, specifically in activated myofibroblasts in the adult mouse heart, slowed LV functional decline and remodeling in a model of pressure overload HF. Our data indicate that ACLY and ACSS2 are necessary for myofibroblast differentiation and persistence. We hypothesize that acetyl-CoA synthesis is necessary for histone acetylation required for activation of myofibroblast genes. Ongoing work will determine the molecular mechanisms of both enzymes in chromatin remodeling and in cardiac injury. In summary, acetyl-CoA producing enzymes are attractive clinical targets to reverse cardiac fibrosis.

Abstract P2025: BET Bromodomain Proteins Are Necessary For TGFβ2-Mediated Myofibroblast Activation

Fibrosis is a key feature of dilated cardiomyopathy (DCM). Stress signaling triggers the activation of quiescent fibroblasts to myofibroblasts. Bromodomain and extraterminal (BET) epigenetic reader proteins integrate stress-responsive signaling. We explored the role of BETs on distinct inflammatory signaling pathways in cardiac fibroblasts. Fibroblasts were isolated from wild type hearts using the Langendorff procedure. Cell cultures were treated with vehicle, the pan-BET inhibitor JQ1, or siRNA targeting Smad3 or Rela (to inhibit TGFβ and NFκB pathways, respectively) followed by TGFβ2 stimulation. Gene expression was assessed by QPCR, α-smooth muscle actin (αSMA) was quantified by western blot, and NFκB transcription was evaluated using a dual luciferase reporter assay. The interaction between BRD4 and SMAD3 was assessed by coimmunoprecipitation (coIP) in immortalized adult mouse cardiac fibroblast cell cultures treated with TGFβ2, JQ1, or vehicle. TGFβ2-induced fibroblast activation was ablated by JQ1 and Smad3 siRNA (reduced expression of Acta2, Col3a1, Postn and αSMA). CoIP revealed a direct interaction between BRD4 and SMAD3 in fibroblasts, which increased 2.2-fold (p<0.05) after TGFβ2 stimulation. By contrast, Rela siRNA alone induced myofibroblast gene expression ( Acta2 , 6.9-fold; Col3a1 , 7.0-fold; Postn , 11.2-fold; p<0.05 vs. vehicle for all), and increased αSMA levels (2.2-fold; p<0.05). This effect of Rela siRNA was similar to that observed with TGFβ2 stimulation. Further, TGFβ2 inhibited NFκB-driven gene transcription (-2.9-fold; p<0.05 vs. vehicle). These data demonstrate that BRD4 is a critical co-activator of TGFβ2-SMAD3 signaling and cardiac fibroblast activation. We also found that NFκB signaling is downregulated by TGFβ2, suggesting that NFκB may modulate inflammatory gene network activation.

Lipoplexes for effective in vitro delivery of microRNAs to adult human cardiac fibroblasts for perspective direct cardiac cell reprogramming

Design of nanocarriers for efficient miRNA delivery can significantly improve miRNA-based therapies. Lipoplexes based on helper lipid, dioleoyl phosphatidylethanolamine (DOPE) and cationic lipid [2-(2,3-didodecyloxypropyl)-hydroxyethyl] ammonium bromide (DE) were formulated to efficiently deliver miR-1 or a combination of four microRNAs (miRcombo) to adult human cardiac fibroblasts (AHCFs). Lipoplexes with amino-to-phosphate groups ratio of 3 (N/P 3) showed nanometric hydrodynamic size (372 nm), positive Z-potential (40 mV) and high stability under storage conditions. Compared to commercial DharmaFECT1 (DF), DE-DOPE/miRNA lipoplexes showed superior miRNA loading efficiency (99 % vs. 64 %), and faster miRNA release (99 % vs. 82 % at 48 h). DE-DOPE/miR-1 lipoplexes showed superior viability (80–100 % vs. 50 %) in AHCFs, a 2-fold higher miR-1 expression and Twinfilin-1 (TWF-1) mRNA downregulation. DE-DOPE/miRcombo lipoplexes significantly enhanced AHCFs reprogramming into induced cardiomyocytes (iCMs), as shown by increased expression of CM markers compared to DF/miRcombo.