Response Of Cardiac Fibroblasts Research Articles

Matrix stiffness controls cardiac fibroblast activation through regulating YAP via AT1 R.

Cardiac fibrosis is a common pathway leading to heart failure and involves continued activation of cardiac fibroblasts (CFs) into myofibroblasts during myocardium damage, causing excessive deposition of the extracellular matrix (ECM) and thus increases matrix stiffness. Increasing evidence has shown that stiffened matrix plays an important role in promoting CF activation and cardiac fibrosis, and several signaling factors mediating CF mechanotransduction have been identified. However, the key molecules that perceive matrix stiffness to regulate CF activation remainto be further explored. Here, we detected significantly increased expression and nuclear localization of Yes-associated protein (YAP) in native fibrotic cardiac tissues. By using mechanically regulated in vitro cell culture models, we found that a stiff matrix-induced high expression and nuclear localization of YAP in CFs, accompanied by enhanced cell activation. We also demonstrated that YAP knockdown decreased fibrogenic response of CFs and that YAP overexpression promoted CF activation, indicating that YAP plays an important role in mediating matrix stiffness-induced CF activation. Further mechanistic studies revealed that the YAP pathway is an important signaling branch downstream of angiotensin II type 1 receptor in CF mechanotransduction. The findings help elucidate the mechanism of fibrotic mechanotransduction and may contribute to the development of new approaches for treating fibrotic diseases.

The ER Unfolded Protein Response Effector, ATF6, Reduces Cardiac Fibrosis and Decreases Activation of Cardiac Fibroblasts.

Activating transcription factor-6 α (ATF6) is one of the three main sensors and effectors of the endoplasmic reticulum (ER) stress response and, as such, it is critical for protecting the heart and other tissues from a variety of environmental insults and disease states. In the heart, ATF6 has been shown to protect cardiac myocytes. However, its roles in other cell types in the heart are unknown. Here we show that ATF6 decreases the activation of cardiac fibroblasts in response to the cytokine, transforming growth factor β (TGFβ), which can induce fibroblast trans-differentiation into a myofibroblast phenotype through signaling via the TGFβ–Smad pathway. ATF6 activation suppressed fibroblast contraction and the induction of α smooth muscle actin (αSMA). Conversely, fibroblasts were hyperactivated when ATF6 was silenced or deleted. ATF6 thus represents a novel inhibitor of the TGFβ–Smad axis of cardiac fibroblast activation.

Natural Compound Library Screening Identifies New Molecules for the Treatment of Cardiac Fibrosis and Diastolic Dysfunction.

Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.

Human Atrial Fibroblast Adaptation to Heterogeneities in Substrate Stiffness.

Fibrosis is associated with aging and many cardiac pathologies. It is characterized both by myofibroblast differentiation and by excessive accumulation of extracellular matrix proteins. Fibrosis-related tissue remodeling results in significant changes in tissue structure and function, including passive mechanical properties. This research area has gained significant momentum with the recent development of new tools and approaches to better characterize and understand the ability of cells to sense and respond to their biophysical environment. We use a novel hydrogel, termed CyPhyGel, to provide an advanced in vitro model of remodeling-related changes in tissue stiffness. Based on light-controlled dimerization of a Cyanobacterial Phytochrome, it enables contactless and reversible tuning of hydrogel mechanical properties with high spatial and temporal resolution. Human primary atrial fibroblasts were cultured on CyPhyGels. After 4 days of culturing on stiff (~4.6 kPa) or soft (~2.7 kPa) CyPhyGels, we analyzed fibroblast cell area and stiffness. Cells grown on the softer substrate were smaller and softer, compared to cells grown on the stiffer substrate. This difference was absent when both soft and stiff growth substrates were combined in a single CyPhyGel, with the resulting cell areas being similar to those on homogeneously stiff gels and cell stiffnesses being similar to those on homogeneously soft substrates. Using CyPhyGels to mimic tissue stiffness heterogeneities in vitro, our results confirm the ability of cardiac fibroblasts to adapt to their mechanical environment, and suggest the presence of a paracrine mechanism that tunes fibroblast structural and functional properties associated with mechanically induced phenotype conversion toward myofibroblasts. In the context of regionally increased tissue stiffness, such as upon scarring or in diffuse fibrosis, such a mechanism could help to prevent abrupt changes in cell properties at the border zone between normal and diseased tissue. The light-tunable mechanical properties of CyPhyGels and their suitability for studying human primary cardiac cells make them an attractive model system for cardiac mechanobiology research. Further investigations will explore the interactions between biophysical and soluble factors in the response of cardiac fibroblasts to spatially and temporally heterogeneous mechanical cues.

Generation of Quiescent Cardiac Fibroblasts Derived from Human Induced Pluripotent Stem Cells.

Myocardial fibrosis is a hallmark of cardiac remodeling, which can progressively lead to heart failure, a leading cause of death worldwide. The effector cells of fibrosis in the heart are cardiac fibroblasts (CFs). There is currently no effective therapeutic strategy clinically available to specifically attenuate maladaptive responses of CFs. Large-scale applications such as high-throughput drug screening are difficult due to the limited availability of human primary CFs, thus limiting the development of future treatments. Here, we describe a robust induction protocol that can be used to generate a scalable, consistent, genetically defined source of quiescent CFs from human induced pluripotent stem cells for cardiac fibrosis modeling, drug discovery, and tissue engineering.

Abstract 257: Cortical Bone Stem Cell-Derived Exosomes Alter Wound Healing Response in Cardiac Fibroblasts and Cardiac Endothelial Cells

Rationale: Post-myocardial infarction (MI) results in remodeling that leads to the development of heart failure. We have previously seen improvements in post-MI wound healing and scar formation as a result of administration of cortical bone stem cell derived exosomes (CBSC exosomes). Objectives: We sought to further elucidate the mechanism through which CBSC exosomes improved scar formation and altered wound healing through in vitro experimentation. We continued to explore the unique characteristics of CBSC exosomes in order to understand why they have anti-fibrotic effects. Methods and Results: We performed migration assays on cardiac endothelial cells and cardiac fibroblasts, and saw alterations in cell migration in both cell types upon treatment with mCBSC CM and mCBSC exosomes. We discovered that migration of CECs was reduced by 20% under the influence of mCBSC CM and mCBSC exosomes, but rescued when exosomes were removed from mCBSC CM. We discovered that the removal of exosomes from mCBSC CM slowed cardiac fibroblast migration by 40%. We saw a decrease in the quantity of pro-fibrotic mRNA in cardiac endothelial cells and cardiac fibroblasts after treatment with mCBSC exosomes. Adult cardiac endothelial cell mRNA levels of VEGFA, Col4A1, and VCAM were reduced nearly two fold from control untreated cell levels following mCBSC exosome treatment. Adult cardiac fibroblast Col1A1, Col3A1, Col4A1, and MMP2 mRNA levels were reduced one-fold from control untreated cell levels following mCBSC exosome treatment. Conclusions: Our findings show that the wound healing induced by CBSC treatment post-MI involves a mechanism altering the migration of endothelial cells, sustaining the migratory capabilities of fibroblasts, and decreasing the production of pro-fibrotic mRNA in cardiac fibroblasts and cardiac endothelial cells.

Abstract 511: Regulation of p53 Protein Levels Drives Activation of Cardiac Fibroblasts in Response to Pressure Overload

Heart disease is accompanied by the accumulation of resident cardiac fibroblasts (CF) that subsequently become myofibroblasts and secrete copious amounts of extracellular matrix (ECM), impeding cardiac function and driving the progression of heart failure. Understanding the mechanisms coordinating CF accumulation and myofibroblast activation may reveal novel therapeutic strategies to block pathologic fibrosis. We recently found that s mall pr oline r ich protein 2b (SPRR2B) drives CF proliferation in response to pathological cues by facilitating MDM2-dependent proteasomal degradation of p53. Surprisingly, although Sprr2b gene deletion or targeted mutation of the USP7/MDM2 interacting domain in mice ( Sprr2b-KO and Sprr2b-USP m , respectively) stimulated the expression of p53-dependent cell cycle arrest genes, mutant animals also developed excessive fibrosis in LV pressure overload. The development of fibrosis in Sprr2b mutant mice was traced primarily to a more robust myofibroblast activation response, providing evidence that CF accumulation and myofibroblast activation may be mutually antagonistic. To investigate the contribution p53 to CF accumulation and fibrosis in mouse heart disease models, we deleted floxed p53 alleles specifically in adult CF using the tamoxifen-inducible Tcf21 MerCreMer mouse line (called p53-CKO). Surprisingly, while p53-CKO animals display exaggerated accumulation of Tcf21 + /PDGFRα + CF in response to left ventricle pressure overload, we observed a biphasic physiological response; initially, p53-CKO animals are resistant to systolic functional decline, only developing more severe fibrosis and functional decline than littermate controls at later time points. Time course studies using primary adult mouse CF revealed that p53 positively correlates with myofibroblast activation, while reduction in p53 levels correlates with accelerated cell cycle and the suppression of myofibroblast activation until the subsequent induction of p16/19 - Rb-mediated cell cycle arrest. Taken together, this study offers detailed insight into the transition of CF from a proliferative to an activated state that may accelerate the development of anti-fibrotic strategies.

The orphan receptor GPRC5B modulates inflammatory and fibrotic pathways in cardiac fibroblasts and mice hearts

Inflammation is a major driver of cardiac remodeling. Cardiac fibroblasts play an integral role in cardiac inflammation, fibrosis and remodeling. The orphan G-protein-coupled-receptor 5B of family C (GPRC5B) has recently been shown to have pro-inflammatory effects in adipocytes via the NFκB-signaling-pathway. Here, we investigated whether GPRC5B is involved in myocardial inflammation and fibrosis.Using neonatal rat cardiac fibroblasts (NRCF) we show that the transcription and the expression of endogenous GPRC5B is induced by stimulation with TNFα and LPS as well as through cyclic mechanical stretch, while the principle pro-fibrotic factor TGFβ has no effect on the GPRC5B expression. Furthermore, we demonstrate that adenoviral overexpression and siRNA-mediated knockdown of GPRC5B in NRCF significantly alters the transcription level of the pro-inflammatory and pro-fibrotic cytokines TNFα, IL-1β, IL-6 and MCP-1, and extracellular matrix-degrading MMP-9 in vitro. Additionally, in adult GPRC5B-transgenic mice the protein expression of collagen-1A1 is decreased and the production of MMP-9 is increased, indicating remodeling of the extracellular matrix in vivo.Our data show that GPRC5B is up-regulated by inflammatory signals and mechanical stress in NRCF, while GPRC5B modulates the inflammatory response of cardiac fibroblasts and the degradation of extracellular matrix-proteins in the mice heart. Thus, our findings are the first to report a novel role of the orphan receptor GPRC5B in fibroblast-driven myocardial inflammation and cardiac remodeling.

From the Literature

From the Literature

Metoprolol Inhibits Profibrotic Remodeling of Epicardial Adipose Tissue in a Canine Model of Chronic Obstructive Sleep Apnea.

BackgroundWhether chronic obstructive sleep apnea (OSA) could promote epicardial adipose tissue (EAT) secretion of profibrotic adipokines, and thereby contribute to atrial fibrosis, and the potential therapeutic effects of metoprolol remain unknown.Methods and ResultsA chronic OSA canine model was established by repeatedly clamping the endotracheal tube for and then reopening it for 4 hours every other day for 12 weeks. In a metoprolol treatment group, metoprolol succinate was administered daily for 12 weeks. The EAT infiltration and left atrial fibrosis were examined. The expressions of adipokines secreted by EAT and hypoxic 3T3‐L1 adipocytes were detected. The changes in collagen synthesis, transforming growth factor‐β1 expression, and cell differentiation and proliferation in cardiac fibroblasts induced by hypoxic 3T3‐L1 adipocyte‐derived conditioned medium were further analyzed. Chronic OSA induced infiltration of EAT into the left atrium. OSA enhanced the profibrotic effect of EAT on the adjacent atrial myocardium. Moreover, OSA induced profibrotic cytokine secretion from EAT. We also found that hypoxia induced adipokine secretion in cultured adipocytes, and the medium conditioned by the hypoxic adipocytes increased collagen and transforming growth factor‐β1 protein expression and cell proliferation of cardiac fibroblasts. More importantly, metoprolol attenuated infiltration of EAT and alleviated the profibrotic effect of EAT by inhibiting adipokine secretion. Metoprolol also inhibited hypoxia‐induced adipokine secretion in adipocytes and thereby blocked the hypoxic adipocyte–derived conditioned medium–induced fibrotic response of cardiac fibroblasts.ConclusionsChronic OSA enhanced the profibrotic effect of EAT on the neighboring atrial myocardium by stimulating the secretion of profibrotic adipokines from EAT, which was significantly attenuated by metoprolol. This study gives insights into mechanisms underlying OSA‐induced atrial fibrillation and also provides experimental evidence for the protective effects of metoprolol.

Exercise promotes a cardioprotective gene program in resident cardiac fibroblasts.

Exercise and heart disease both induce cardiac remodeling, but only disease causes fibrosis and compromises heart function. The cardioprotective benefits of exercise have been attributed to changes in cardiomyocyte physiology, but the impact of exercise on cardiac fibroblasts (CFs) is unknown. Here, RNA-sequencing reveals rapid divergence of CF transcriptional programs during exercise and disease. Among the differentially expressed programs, NRF2-dependent antioxidant genes - including metallothioneins (Mt1 and Mt2) - are induced in CFs during exercise and suppressed by TGF-β/p38 signaling in disease. In vivo, mice lacking Mt1/2 exhibit signs of cardiac dysfunction in exercise, including cardiac fibrosis, vascular rarefaction, and functional decline. Mechanistically, exogenous MTs derived from fibroblasts are taken up by cultured cardiomyocytes, reducing oxidative damage-dependent cell death. Importantly, suppression of MT expression is conserved in human heart failure. Taken together, this study defines the acute transcriptional response of CFs to exercise and disease and reveals a cardioprotective mechanism that is lost in disease.

Integrin \u03b1\u03bd\u03b25 in vitro inhibition limits pro-fibrotic response in cardiac fibroblasts of spontaneously hypertensive rats

BackgroundTo date the TGF-β1 activation mediated by integrin ανβ5 during fibrosis is well-known. This process has been shown also in the heart, where cardiac fibroblasts (CF) differentiate into α-smooth muscle actin (α-SMA)-positive myofibroblasts (MyoFB). Here, we studied the effects on CF, isolated by spontaneously hypertensive rats (SHR), of integrin ανβ5 inhibition in MyoFB differentiation.MethodsStaining and immunohistochemistry were performed on rat cardiac tissue. CF were isolated by enzymatic digestion from SHR (SHR-CF) and normotensive WKY (WKY-CF) rat hearts and then treated for in vitro evaluation.ResultsSHR heart tissues revealed a higher TGF-β1 expression vs. WKY samples. SHR-CF showed an enhanced SMAD2/3 activation and an up-regulated expression of α-SMA, a typical MyoFB marker, especially after TGF-β1 treatment. Immunostaining on cardiac tissues revealed a higher expression of integrin ανβ5 in SHR vs. WKY rat hearts. In vitro results confirmed the up-regulation of integrin ανβ5 expression in SHR-CF at basal condition and after TGF-β1 treatment, in comparison with WKY-CF. Inhibition of integrin ανβ5 by cilengitide treatment led a decreased expression of ανβ5, collagen I, and α-SMA in SHR-CF vs. WKY-CF, resulting in a diminished differentiation of CF into MyoFB. Taking together, results suggested that SHR-CF are more susceptible to TGF-β1, showing an up-regulated activation of SMAD2/3 signaling, and an increased ανβ5, α-SMA, and collagen I expression. Hypertension stimulus promoted an up-regulation of integrin ανβ5 on SHR cardiac tissue and its in vitro inhibition reverted pro-fibrotic events of SHR-CF.ConclusionInhibition of integrin ανβ5 exerted by cilengitide strongly diminished SHR-CF differentiation into detrimental MyoFB. So, integrin ανβ5 might be considered a novel therapeutic target and cilengitide an effective pharmacological tool to limit the progression of hypertension-induced cardiac fibrosis.

IFN-β Plays Both Pro- and Anti-inflammatory Roles in the Rat Cardiac Fibroblast Through Differential STAT Protein Activation.

Cardiac fibroblasts (CFs) contribute to theinflammatory response to tissue damage, secreting both pro- and anti-inflammatory cytokines and chemokines. Interferon beta (IFN-β) induces the phosphorylation of signal transducer and activator of transcription (STAT) proteins through the activation of its own receptor, modulating the secretion of cytokines and chemokines which regulate inflammation. However, the role of IFN-β and STAT proteins in modulating the inflammatory response of CF remains unknown. CF were isolated from adult male rats and subsequently stimulated with IFN-β to evaluate the participation of STAT proteins in secreting chemokines, cytokines, cell adhesion proteins expression and in their capacity to recruit neutrophils. In addition, in CF in which the TRL4 receptor was pre-activated, the effect of INF-β on the aforementioned responses was also evaluated. Cardiac fibroblasts stimulation with IFN-β showed an increase in STAT1, STAT2, and STAT3 phosphorylation. IFN-β stimulation through STAT1 activation increased proinflammatory chemokines MCP-1 and IP-10 secretion, whereas IFN-β induced activation of STAT3 increased cytokine secretion of anti-inflammatory IL-10. Moreover, in TLR4-activated CF, IFN-β through STAT2 and/or STAT3, produced an anti-inflammatory effect, reducing pro-IL-1β, TNF-α, IL-6, MCP-1, and IP-10 secretion; and decreasing neutrophil recruitment by decreasing ICAM-1 and VCAM-1 expression. Altogether, our results indicate that IFN-β exerts both pro-inflammatory and anti-inflammatory effects in non-stimulated CF, through differential activation of STAT proteins. When CF were previously treated with an inflammatory agent such as TLR-4 activation, IFN-β effects were predominantly anti-inflammatory.

Nogo-C regulates post myocardial infarction fibrosis through the interaction with ER Ca2+ leakage channel Sec61\u03b1 in mouse hearts

Cardiac fibrosis is an independent risk factor for heart failure and even the leading cause of death in myocardial infarction patients. However, molecular mechanisms associated with the pathogenesis of cardiac fibrosis following myocardial infarction are not yet fully understood. Nogo-C protein ubiquitously expresses in tissues including in the heart. Our previous study found that Nogo-C regulated cardiomyocyte apoptosis during myocardial infarction. In the present study, we found that Nogo-C was upregulated in fibrotic hearts after myocardial infarction and in Ang II- or TGF-β1-stimulated cardiac fibroblasts. Overexpression of Nogo-C in cardiac fibroblasts increased expression of pro-fibrogenic proteins, while knockdown of Nogo-C inhibited the fibrotic responses of cardiac fibroblasts to Ang II- or TGF-β1 stimulation. Functionally, Nogo-C deficiency suppressed pro-fibrogenic proteins in post-myocardial infarction hearts and ameliorated post-myocardial infarction cardiac function. Mechanistically, we found that Nogo-C increased intracellular Ca2+ concentration and buffering Ca2+ totally abolished Nogo-C-induced fibrotic responses. Moreover, overexpression of Nogo-C caused increased Sec61α, the Ca2+ leakage channel on endoplasmic reticulum membrane. Nogo-C interacted with Sec61α on endoplasmic reticulum and stabilized Sec61α protein by inhibiting its ubiquitination. Inhibition or knockdown of Sec61α blocked Nogo-C-induced increase of cytosolic Ca2+ concentration and inhibited Nogo-C- and TGF-β1-induced fibrotic responses in cardiac fibroblasts, suggesting that Nogo-C regulates cardiac fibrosis through interacting with Sec61α to mediate the Ca2+ leakage from endoplasmic reticulum. Thus, our results reveal a novel mechanism underlying cardiac fibrosis following myocardial infarction, and provide a therapeutic strategy for cardiac remodeling related heart diseases.

Up-regulation of NOX1/NADPH oxidase following drug-induced myocardial injury promotes cardiac dysfunction and fibrosis.

Cardiac fibrosis is a common feature in failing heart and therapeutic strategy to halt the progression of fibrosis is highly needed. We here report on NOX1, a non-phagocytic isoform of superoxide-producing NADPH oxidase, which promotes cardiac fibrosis in a drug-induced myocardial injury model. A single-dose administration of doxorubicin (DOX) elicited cardiac dysfunction accompanied by increased production of reactive oxygen species and marked elevation of NOX1 mRNA in the heart. In mice deficient in Nox1 (Nox1-/Y), cardiac functions were well retained and overall survival was significantly improved. However, increased level of serum creatine kinase was equivalent to that of wild-type mice (Nox1+/Y). At 4 days after DOX treatment, severe cardiac fibrosis accompanied by increased hydroxyproline content and activation of matrix metalloproteinase-9 was demonstrated in Nox1+/Y, but it was significantly attenuated in Nox1-/Y. When H9c2 cardiomyocytes were exposed to their homogenate, a dose-dependent increase in NOX1 mRNA was observed. Up-regulation of NOX1 mRNA in H9c2 co-incubated with their homogenate was abolished in the presence of TAK242, a TLR4 inhibitor. When isolated cardiac fibroblasts were exposed to H9c2 homogenates, increased proliferation and up-regulation of collagen 3a1 mRNA were demonstrated. These changes were significantly attenuated in cardiac fibroblasts exposed to homogenates from H9c2 harboring disrupted Nox1. These findings suggest that up-regulation of NOX1 following cellular damage promotes cardiac dysfunction and fibrosis by aggravating the pro-fibrotic response of cardiac fibroblasts. Modulation of the NOX1/NADPH oxidase signaling pathway may be a novel therapeutic strategy for preventing heart failure after myocardial injury.

Serum-glucocorticoid-regulated kinase 1 contributes to mechanical stretch-induced inflammatory responses in cardiac fibroblasts.

Excessive mechanical stretch induces production of proinflammatory mediators in cardiac fibroblasts, which could act as inflammatory supporter cells in heart failure. Accumulation evidence and our previous studies suggest that serum-glucocorticoid-regulated kinase 1 (SGK1) contributes to cardiac remodeling and fibrosis, development of heart failure. However, the role and mechanism of SGK1 in mechanical stretch-induced inflammation of cardiac fibroblasts remain unclear. Here, cardiac fibroblasts isolated from wild-type (WT) and SGK1 knockout (SGK1-/-) mice were stimulated by 18% cyclic stretch, under static condition as the control. The results showed that mechanical stretch increased SGK1 expression and activation in WT cardiac fibroblasts but not its isoform, SGK2 or SGK3 expression. Bio-Plex array revealed hyperstretch could enhance chemokines release in WT cardiac fibroblasts, but SGK1 knockout significantly attenuated chemokines production through blocking activation of nuclear factor-kappa B (NF-κB). Moreover, supernatants from WT cardiac fibroblasts subjected to hyperstretch promoted macrophage migration, enhanced expression of macrophage-derived profibrotic mediators, whereas supernatants from SGK1 deficiency suppressed these effects. Although SGK1 did not directly affect mechanical stretch-induced myofibroblast differentiation, SGK1 activation of cardiac fibroblasts facilitated myofibroblast differentiation through the upregulation of the profibrotic mediators secreted by macrophages. These results suggest that SGK1 may play a critical role in the inflammatory cascade of cardiac fibroblasts triggered by mechanical stretch; SGK1 could be used as a potential target for treatment of cardiac fibrosis and heart failure.

Abstract 108: Exercise Promotes a Cardioprotective Gene Program in Resident Cardiac Fibroblasts

Exercise and disease both induce hypertrophic cardiac growth, but only disease results in fibrosis and compromised heart function. Transcriptional profiling of resident cardiac fibroblasts (CFs), the primary cellular source of fibrosis, was used to define the gene expression programs (GEP) underlying this divergent functional outcome. Bioinformatic analyses revealed distinct transcriptional responses to exercise and disease, including induction of Rho- and SRF-dependent remodeling genes in disease and NRF2-dependent antioxidant genes in exercise. The expression of a number of antioxidant genes, including metallothioneins (Mt1 and Mt2), are specifically maintained in CFs after exercise and lost in disease. Mice lacking Mt1/2 show signs of cardiac dysfunction after exercise, including cardiac fibrosis, vascular rarefaction, and reduced heart function. Importantly, Mt levels are also reduced in human heart failure (HF) patients, suggesting a potentially conserved cardioprotective role in humans. Non-canonical TGF-β1-mediated p38-MAPK signaling has previously been implicated in HF, therefore we tested the role of p38 signaling in Mt regulation. Pharmacological inhibition of p38 in human HF fibroblasts restores Mt1 and Mt2 expression and blunts the pathological fibroblast phenotype. Taken together, our study defines the transcriptional response of CFs to exercise and disease and reveals a cardioprotective mechanism that is lost in disease.

CYP2J2 metabolites, epoxyeicosatrienoic acids, attenuate Ang II-induced cardiac fibrotic response by targeting Gα12/13

The arachidonic acid-cytochrome P450 2J2-epoxyeicosatrienoic acid (AA-CYP2J2-EET) metabolic pathway has been identified to be protective in the cardiovascular system. This study explored the effects of the AA-CYP2J2-EET metabolic pathway on cardiac fibrosis from the perspective of cardiac fibroblasts and underlying mechanisms. In in vivo studies, 8-week-old male CYP2J2 transgenic mice (aMHC-CYP2J2-Tr) and littermates were infused with angiotensin II (Ang II) or saline for 2 weeks. Results showed that CYP2J2 overexpression increased EET production. Meanwhile, impairment of cardiac function and fibrotic response were attenuated by CYP2J2 overexpression. The effects of CYP2J2 were associated with reduced activation of the α subunits of G12 family G proteins (Gα12/13)/RhoA/Rho kinase (ROCK) cascade and elevation of the NO/cyclic guanosine monophosphate (cGMP) level in cardiac tissue. In in vitro studies, cardiac fibroblast activation, proliferation, migration, and collagen production induced by Ang II were associated with activation of the Gα12/13/RhoA/ROCK pathway, which was inhibited by exogenous 11,12-EET. Moreover, silencing of Gα12/13 or RhoA exerted similar effects as 11,12-EET. Furthermore, inhibitory effects of 11,12-EET on Gα12/13 were blocked by NO/cGMP pathway inhibitors. Our findings indicate that enhancement of the AA-CYP2J2-EET metabolic pathway by CYP2J2 overexpression attenuates Ang II-induced cardiac dysfunction and fibrosis by reducing the fibrotic response of cardiac fibroblasts by targeting the Gα12/13/RhoA/ROCK pathway via NO/cGMP signaling.

Betulinic acid prevents high glucose‑induced expression of extracellular matrix protein in cardiac fibroblasts by inhibiting the TGF‑β1/Smad signaling pathway.

The proliferation and differentiation of cardiac fibroblasts (CFs) is central to cardiac fibrosis. Betulinic acid (BA) is an active compound isolated from the bark of the birch tree Betula spp. (Betulaceae) and has been shown to attenuate hepatic fibrosis. However, the effect of BA on the high glucose‑induced fibrosis response in CFs remains to be elucidated, therefore, the present study investigated the effect of BA on high glucose‑induced CFs and examined the possible mechanism underlying the effect of BA on CF transdifferentiation. CFs were pre‑incubated with various concentrations of BA for 24h and then stimulated with high glucose (25mM) for various times. Cell proliferation was evaluated using an MTT assay. The mRNA expression levels of α‑smooth muscle actin (SMA) and transforming growth factor (TGF)‑β1 were determined using reverse transcription‑quantitative polymerase chain reaction analysis. The protein expression levels of α‑SMA, collagen I, collagen III, fibronectin, TGF‑β1, small mothers against decapentaplegic (Smad)2/3, phosphorylated (p)‑Smad2 and p‑Smad3 and were detected using western blot analysis. The data revealed that BA attenuated the CF proliferation and myofibroblast differentiation induced by high glucose. In addition, BA inhibited the expression of extracellular matrix (ECM) in the CFs induced by high glucose. It was also found that BA inhibited the high glucose‑induced phosphorylation of Smad2/3 in the CFs. Taken together, BA suppressed the high glucose‑induced increase in the proliferation of CFs and expression of ECM via inhibition of the TGF‑β1/Smad signaling pathway. Thus, BA may offer therapeutic potential towards the treatment of cardiac fibrosis.

Tissue transglutaminase induction in the pressure-overloaded myocardium regulates matrix remodelling.

Tissue transglutaminase (tTG) is induced in injured and remodelling tissues, and modulates cellular phenotype, while contributing to matrix cross-linking. Our study tested the hypothesis that tTG may be expressed in the pressure-overloaded myocardium, and may regulate cardiac function, myocardial fibrosis and chamber remodelling. In order to test the hypothesis, wild-type and tTG null mice were subjected to pressure overload induced through transverse aortic constriction. Moreover, we used isolated cardiac fibroblasts and macrophages to dissect the mechanisms of tTG-mediated actions. tTG expression was upregulated in the pressure-overloaded mouse heart and was localized in cardiomyocytes, interstitial cells, and in the extracellular matrix. In contrast, expression of transglutaminases 1, 3, 4, 5, 6, 7 and FXIII was not induced in the remodelling myocardium. In vitro, transforming growth factor (TGF)-β1 stimulated tTG synthesis in cardiac fibroblasts and in macrophages through distinct signalling pathways. tTG null mice had increased mortality and enhanced ventricular dilation following pressure overload, but were protected from diastolic dysfunction. tTG loss was associated with a hypercellular cardiac interstitium, reduced collagen cross-linking, and with accentuated matrix metalloproteinase (MMP)2 activity in the pressure-overloaded myocardium. In vitro, tTG did not modulate TGF-β-mediated responses in cardiac fibroblasts; however, tTG loss was associated with accentuated proliferative activity. Moreover, when bound to the matrix, recombinant tTG induced synthesis of tissue inhibitor of metalloproteinases (TIMP)-1 through transamidase-independent actions. Following pressure overload, endogenous tTG mediates matrix cross-linking, while protecting the remodelling myocardium from dilation by exerting matrix-preserving actions.