Signaling In Cardiac Fibroblasts Research Articles

SFRP2 activates Wnt/β-catenin signaling in cardiac fibroblasts: differential roles in cell growth, energy metabolism, and extracellular matrix remodeling.

Secreted Frizzled-related protein 2 (sFRP2) plays a key role in chronic fibrosis after myocardial infarction and in heart failure. The aim of this study was to elucidate the mechanisms through which sFRP2 may regulate the growth and extracellular matrix (ECM) remodeling of adult mouse cardiac fibroblasts (CFs). We found that sFRP2 activates CFs in part through canonical Wnt/β-catenin signaling, as evidenced by increased expression of Axin2 and Wnt3a, but not Wnt5a, as well as accumulation of nuclear β-catenin. In response to sFRP2, CFs exhibited robust cell proliferation associated with increased glucose consumption and lactate production, a phenomenon termed "the Warburg effect" in oncology. The coupling between CF expansion and anaerobic glycolysis is marked by upregulation of glyceraldehyde-3-phosphate dehydrogenase and tissue-nonspecific alkaline phosphatase. In conjunction with these phenotypic changes, CFs accelerated ECM remodeling through upregulation of expression of the matrix metalloproteinase (MMP) 1 and MMP13 genes, two members of the collagenase subfamily, and enzyme activities of MMP2 and MMP9, two members of the gelatinase subfamily. Consistent with the induction of multiple MMPs possessing collagenolytic activities, the steady-state level of collagen type 1 in CF-spent medium was reduced by sFRP2. Analysis of non-CF cell types revealed that the multifaceted effects of sFRP2 on growth control, glucose metabolism, and ECM regulation are largely restricted to CFs and highly sensitive to Wnt signaling perturbation. The study provides a molecular framework on which the functional versatility and signaling complexity of sFRP2 in cardiac fibrosis may be better defined.

Regulation of fibronectin gene expression in cardiac fibroblasts by scleraxis.

The glycoprotein fibronectin is a key component of the extracellular matrix. By interacting with numerous matrix and cell surface proteins, fibronectin plays important roles in cell adhesion, migration and intracellular signaling. Up-regulation of fibronectin occurs in tissue fibrosis, and previous studies have identified the pro-fibrotic factor TGFβ as an inducer of fibronectin expression, although the mechanism responsible remains unknown. We have previously shown that a key downstream effector of TGFβ signaling in cardiac fibroblasts is the transcription factor scleraxis, which in turn regulates the expression of a wide variety of extracellular matrix genes. We noted that fibronectin expression tracked closely with scleraxis expression, but it was unclear whether scleraxis directly regulated the fibronectin gene. Here, we report that scleraxis acts via two E-box binding sites in the proximal human fibronectin promoter to govern fibronectin expression, with the second E-box being both sufficient and necessary for scleraxis-mediated fibronectin expression to occur. A combination of electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that scleraxis interacted to a greater degree with the second E-box. Over-expression or knockdown of scleraxis resulted in increased or decreased fibronectin expression, respectively, and scleraxis null mice presented with dramatically decreased immunolabeling for fibronectin in cardiac tissue sections compared to wild-type controls. Furthermore, scleraxis was required for TGFβ-induced fibronectin expression: TGFβ lost its ability to induce fibronectin expression following scleraxis knockdown. Together, these results demonstrate a novel and required role for scleraxis in the regulation of cardiac fibroblast fibronectin gene expression basally or in response to TGFβ.

Angiotensin II‐mediated Mitochondrial Ca2+ Uptake and Superoxide Generation Activate Proliferative Pathway in Neonatal Cardiac Fibroblasts

Cardiac fibroblasts (CFs) are the most prevalent cell type in the heart in addition to cardiomyocytes and play key roles in regulating myocardial physiological function and pathophysiological remodeling. Clinical observations and basic research data strongly suggest that CFs can respond to various stimuli including, angiotensin II (AT‐II), the levels of which are increased in the remodeling heart and participates in remodeling of the failing heart by AT‐II‐mediated pathological CF proliferation. It has been shown that CF proliferation may occur via enhanced production of reactive oxygen species (ROS), but the detailed signal transduction remains unclear. We previously reported that the enhancement of mitochondrial Ca2+ uptake by mitochondrial Ca2+ uniporter (MCU) induces mitochondrial superoxide (mtSO) generation in cardiomyocytes. Therefore, we hypothesize that Ang‐II stimulation enhances mitochondrial Ca2+‐ induced mtSO generation in CFs, which can activate ROS‐dependent proliferation signaling in CFs. First, using a mitochondria‐targeted Ca2+ biosensor, we found that AT‐II (≥1 μM) stimulation induces significant mitochondrial Ca2+ uptake in response to the Ca2+ release from the endoplasmic reticulum in neonatal rat CFs (NCF). In addition, AT‐II stimulation increases the mtSO levels detected by a mtSO indicator MitoSOX Red. These effects were significantly blocked by the pretreatment of an AT‐receptor antagonist losartan. Next, we confirmed that AT‐II application activates proliferative pathway, including ERK1/2, p38 and JNK1/2 in time‐dependent manner, which was abolished by losartan pretreatment. Lastly, pretreatment of a mitochondria‐targeted antioxidant, Mito‐tempo also significantly inhibited AT‐II‐mediated activation of the proliferative pathway without changing the AT‐II‐induced the mitochondrial Ca2+ uptake profile. Our results indicate that mtSO generation induced by mitochondrial Ca2+ accumulation works as a stimulator of the Ang II–induced proliferative pathway in cardiac fibroblasts.Support or Funding InformationThis work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).

The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy.

The heart responds to pathological overload through myocyte hypertrophy. Here we show that this response is regulated by cardiac fibroblasts via a paracrine mechanism involving plasma membrane calcium ATPase 4 (PMCA4). Pmca4 deletion in mice, both systemically and specifically in fibroblasts, reduces the hypertrophic response to pressure overload; however, knocking out Pmca4 specifically in cardiomyocytes does not produce this effect. Mechanistically, cardiac fibroblasts lacking PMCA4 produce higher levels of secreted frizzled related protein 2 (sFRP2), which inhibits the hypertrophic response in neighbouring cardiomyocytes. Furthermore, we show that treatment with the PMCA4 inhibitor aurintricarboxylic acid (ATA) inhibits and reverses cardiac hypertrophy induced by pressure overload in mice. Our results reveal that PMCA4 regulates the development of cardiac hypertrophy and provide proof of principle for a therapeutic approach to treat this condition.

A computational model of cardiac fibroblast signaling predicts context-dependent drivers of myofibroblast differentiation

Cardiac fibroblasts support heart function, and aberrant fibroblast signaling can lead to fibrosis and cardiac dysfunction. Yet how signaling molecules drive myofibroblast differentiation and fibrosis in the complex signaling environment of cardiac injury remains unclear. We developed a large-scale computational model of cardiac fibroblast signaling in order to identify regulators of fibrosis under diverse signaling contexts. The model network integrates 10 signaling pathways, including 91 nodes and 134 reactions, and it correctly predicted 80% of independent previous experiments. The model predicted key fibrotic signaling regulators (e.g. reactive oxygen species, tissue growth factor β (TGFβ) receptor), whose function varied depending on the extracellular environment. We characterized how network structure relates to function, identified functional modules, and predicted cross-talk between TGFβ and mechanical signaling, which was validated experimentally in adult cardiac fibroblasts. This study provides a systems framework for predicting key regulators of fibroblast signaling across diverse signaling contexts.

Fibrosis of the Neonatal Mouse Heart After Cryoinjury Is Accompanied by Wnt Signaling Activation and Epicardial-to-Mesenchymal Transition.

BackgroundThe adult mammalian heart responds to cardiac injury by formation of persistent fibrotic scar that eventually leads to heart failure. In contrast, the neonatal mammalian heart reacts to injury by the development of transient fibrotic tissue that is eventually replaced by regenerated cardiomyocytes. How fibrosis occurs in the neonatal mammalian heart remains unknown. To start elucidating the molecular underpinnings of neonatal cardiac fibrosis, we investigated Wnt signaling in the neonatal heart after cryoinjury.Methods and ResultsUsing expression of the Wnt target gene Axin2 as an indicator of Wnt/β‐catenin signaling activation, we discovered that epicardial cells in the ventricles are responsive to Wnt in the uninjured neonatal heart. Lineage‐tracing studies of these Wnt‐responsive epicardial cells showed that they undergo epithelial‐to‐mesenchymal transition and infiltrate into the subepicardial space and exhibit fibroblast phenotypes after injury. In addition, we showed that—similar to adult ischemic injury—neonatal cryoinjury results in activation of Wnt signaling in cardiac fibroblasts near injured areas. Furthermore, through in situ hybridization of all 19 Wnt ligands in injured neonatal hearts, we observed upregulation of Wnt ligands (Wnt2b, Wnt5a, and Wnt9a) that had not been implicated in the adult cardiac injury response.ConclusionsThese results demonstrate that cryoinjury in neonatal heart leads to the formation of fibrotic tissue that involves Wnt‐responsive epicardial cells undergoing epithelial‐to‐mesenchymal transition to give rise to fibroblasts and activation of Wnt signaling in resident cardiac fibroblasts.

Abstract 11340: The Plasma Membrane Calcium ATPase 4 Signaling in Cardiac Fibroblasts Mediates Cardiomyocyte Hypertrophy (Best of Basic Science Abstract)

Plasma membrane calcium ATPase 4 (PMCA4) is a ubiquitously expressed Ca 2+ pump that is important in mediating molecular signaling in the heart. Here we show a novel role of PMCA4 in cardiac fibroblasts in controlling cardiac hypertrophy. We generated three PMCA4 knockout mouse strains: i) PMCA4 global knockout (PMCA4 -/- ); ii) PMCA4 cardiomyocyte-specific knockout (PMCA4 cko ); iii) PMCA4 fibroblast-specific knockout (PMCA4 fko ). Following transverse aortic constriction (TAC) for 5 weeks, PMCA4 -/- and PMCA4 fko mice displayed a significantly reduced hypertrophy compared to control mice. In contrast, the PMCA4 cardiac specific knockout (PMCA4 cko ) mice did not show any protective effect following TAC, prompting us to hypothesise that the protective effect might be due to PMCA4 ablation in fibroblasts. Microarray analysis revealed a ~100 fold upregulation of secreted frizzled-related protein 2 (sFRP2) in PMCA4 -/- fibroblasts, which was further confirmed by qRT-PCR and Western blot. sFRP2 is a potent inhibitor of the Wnt/β-catenin pathway. We then cultured wild type (WT) cardiomyocytes with conditioned medium from either PMCA4 -/- or WT fibroblasts. In response to phenylephrine stimulation, cardiomyocytes cultured in PMCA4 -/- fibroblast–conditioned medium displayed 88% less hypertrophy than those cultured in WT fibroblast–conditioned medium (P<0.01). Mechanistically, PMCA4 -/- fibroblasts showed a significant elevation in NFκB activity, a transcription factor that regulates sFRP2 expression. Inhibition of NFκB activity significantly reduced the sFRP2 expression in PMCA4 -/- fibroblasts to a level comparable to WT expression. A chemical library screen identified a novel PMCA4 inhibitor, aurintricarboxylic acid (ATA). ATA treatment enhanced sFRP2 expression in the mouse heart. Importantly, ATA inhibited and reversed TAC-induced cardiac hypertrophy in mice, demonstrating its efficacy in both preventive and therapeutic strategies. In conclusion, PMCA4-mediated signaling in cardiac fibroblasts plays a key role in controlling cardiac hypertrophy. Cardiac fibroblasts lacking PMCA4 produce higher levels of sFRP2, which protects the neighbouring cardiomyocytes. Finally, PMCA4 is a potential target for cardiac hypertrophy treatment.

Abstract 13837: Adrenergic Receptors β2 and β3 Transduce Differential Signals in Cardiac Fibroblasts

Background: Cardiac fibroblasts (CFs) are the most prevalent cell types in heart and play important roles in cardiac remodeling. While the roles of β-adrenergic receptor (βAR) signaling in cardiomyocytes (CMs) are well characterized, those in CFs remain to be elusive due to lack of convenient method to assess those signaling. There are three subtypes of, βAR β1, β2, β3 and β2AR is reported to be expressed in CFs by which enhances cell proliferation and production of inflammatory cytokines. Clinical efficacy of non-selective β blocker carvedilol for heart failure (HF) surpasses that of β1 selective blocker metoprolol, suggesting critical roles of β2 and β3AR in the pathogenesis of HF. Objective: To elucidate the signaling downstream βARs in CFs in heart. Methods and Results: Caveolae is an important microdomain for signal transduction, such as βAR, present in CMs or CFs. To elucidate βAR signaling of caveolae in CFs, we generated a fusion protein composed of phospholamban (PLN) and caveolin3 (Cav3) representing PKA activation as phosphorylation at S16 of PLN and CaMKII as that at T17 in caveolae. Thus, activation of PKA or CaMKII is detectable by anti-phospho-S16 or T17 antibody, respectively. In neonatal rat CFs (NRCFs) infected PLN-Cav3 adenovirus, stimulation by isoproterenol (ISO) led to enhanced phosphorylation of both S16 and T17, suggesting PKA and CaMKII activation in caveolae of CFs. RT-PCR analyses showed β2AR and β3AR were present in NRCFs. Stimulation with β2AR selective agonists activated both PKA and CaMKII, while β3AR elicited solely PKA activation, analyzed by using β3AR selective agonist/antagonist. In addition, in order to examine the significance of βAR stimulation for heart failure, we administered ISO continuously for two weeks in β2ARKO mice. As a result, fibrosis was suppressed in β2ARKO mice compared with wild-type mice (0.35% vs 2.37%, p<0.05) suggesting critical roles of β2AR in development of cardiac fibrosis caused by βAR stimulation in mice. Conclusions: Both β2 and β3AR are expressed in NRCFs and transduce distinct signaling and β2AR selective stimulation elicit development of cardiac fibrosis via activation of CaMKII signaling. Thus, selective βAR regulation could be potential novel anti-fibrotic therapeutics in HF.

P63RhoGEF regulates auto- and paracrine signaling in cardiac fibroblasts

Cardiac remodeling, a hallmark of heart disease, is associated with intense auto- and paracrine signaling leading to cardiac fibrosis. We hypothesized that the specific mediator of Gq/11-dependent RhoA activation p63RhoGEF, which is expressed in cardiac fibroblasts, plays a role in the underlying processes. We could show that p63RhoGEF is up-regulated in mouse hearts subjected to transverse aortic constriction (TAC). In an engineered heart muscle model (EHM), p63RhoGEF expression in cardiac fibroblasts increased resting and twitch tensions, and the dominant negative p63ΔN decreased both. In an engineered connective tissue model (ECT), p63RhoGEF increased tissue stiffness and its knockdown as well as p63ΔN reduced stiffness. In 2D cultures of neonatal rat cardiac fibroblasts, p63RhoGEF regulated the angiotensin II (Ang II)-dependent RhoA activation, the activation of the serum response factor, and the expression and secretion of the connective tissue growth factor (CTGF). All these processes were inhibited by the knockdown of p63RhoGEF or by p63ΔN likely based on their negative influence on the actin cytoskeleton. Moreover, we show that p63RhoGEF also regulates CTGF in engineered tissues and correlates with it in the TAC model. Finally, confocal studies revealed a closely related localization of p63RhoGEF and CTGF in the trans-Golgi network.

Abstract 250: Elucidating the Role of Platelet Derived Growth Factor Receptor Alpha Signaling in Cardiac Fibroblasts

Cardiac fibrosis is a major component of heart disease and is a hallmark of decreased cardiac function. Currently, there are no treatments that attenuate fibrosis directly. This major hurdle can be overcome by targeting the resident fibroblast. Preliminary data demonstrates that loss of PDGFRα expression in the adult cardiac fibroblast lineage results in loss of over half of resident fibroblasts. A time course experiment revealed that in as little as 4 days after PDGFRα gene deletion fibroblast loss can observed. Based on the basal level of fibroblast proliferation (0.8%+/-0.9, i.e. 4 of 398 cells), we hypothesize that PDGFRα signaling is essential for fibroblast maintenance and that fibroblasts undergo rapid turnover. We have begun to elucidate which downstream signals of PDGFRα are involved the different roles of the fibroblast. Using a PDGFRα-dependent-PI3K-deficient mouse model, preliminary data indicates that PDGFRα-dependent PI3K signaling is involved in this cell survival response. Future studies will investigate cardiac fibroblast maintenance signals by determining which cell types secrete PDGF ligands. We will also investigate the role of PDGFRα signaling after myocardial infarction. Our lab has genetic tools that enable us to follow fibroblasts after injury, and we have determined both the number of proliferating fibroblasts at different time points, as well as the fraction of fibroblasts that make up the total population of proliferating cells after LAD ligation. Our preliminary data in control hearts shows that fibroblasts reach their peak of proliferation within a week after infarction, although they remain one of the most proliferative cell types as long as three weeks after induction. Our studies will illuminate the role of the fibroblast in tissue homeostasis and after infarction and identify how these cells contribute to overall cardiovascular function and delineate the fine balance between the essential and detrimental functions of the fibroblast.

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

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

PKC δ and βII regulate angiotensin II-mediated fibrosis through p38: a mechanism of RV fibrosis in pulmonary hypertension.

Pulmonary hypertension (PH) eventually leads to right ventricular (RV) fibrosis and dysfunction that is associated with increased morbidity and mortality. Although angiotensin II plays an important role in RV remodeling associated with hypoxic PH, the molecular mechanisms underlying RV fibrosis in PH largely remain unresolved. We hypothesized that PKC-p38 signaling is involved in RV collagen accumulation in PH and in response to angiotensin II stimulation. Adult male Sprague-Dawley rats were exposed to 3 wk of normoxia or hypoxia (10% FiO2 ) as a model of PH. Hypoxic rats developed RV hypertrophy and fibrosis associated with an increase in PKC βII and δ protein expression and p38 dephosphorylation in freshly isolated RV cardiac fibroblasts. Further mechanistic studies were performed in cultured primary cardiac fibroblasts stimulated with angiotensin II, a key activator of ventricular fibrosis in PH. Angiotensin II induced a reduction in p38 phosphorylation that was attenuated following chemical inhibition of PKC βII and δ. Molecular and chemical inhibition of PKC βII and δ abrogated angiotensin II-induced cardiac fibroblast proliferation and collagen deposition in vitro. The effects of PKC inhibition on proliferation and fibrosis were reversed by chemical inhibition of p38. Conversely, constitutive activation of p38 attenuated angiotensin II-induced increase of cardiac fibroblast proliferation and collagen accumulation. PKC βII- and δ-dependent inactivation of p38 regulates cardiac fibroblast proliferation and collagen deposition in response to angiotensin II, which suggests that the PKC-p38 signaling in cardiac fibroblasts may be involved and important in the pathophysiology of RV fibrosis in PH.

SLC41A1 knockdown inhibits angiotensin II-induced cardiac fibrosis by preventing Mg2+ efflux and Ca2+ signaling in cardiac fibroblasts

Na+/Mg2+ exchanger plays an important role in cardiovascular system, but the molecular mechanisms still largely remain unknown. The Solute Carrier family 41A1 (SLC41A1), a novel Mg2+ transporter, recently was found to function as Na+/Mg2+ exchanger, which mainly regulates the intracellular Mg2+ ([Mg2+]i) homeostasis. Our present studies were designed to investigate whether SLC41A1 impacts on the fibrogenesis of cardiac fibroblasts under Ang II stimulation. Our results showed that quinidine, a prototypical inhibitor of Na+/Mg2+ exchanger, inhibited Ang II-induced cardiac fibrosis via attenuating the overexpression of vital biomarkers of fibrosis, including connective tissue growth factor (CTGF), fibronectin (FN) and α-smooth muscle actin (α-SMA). In addition, quinidine also decreased the Ang II-mediated elevation of concentration of intracellular Ca2+ ([Ca2+]i) and extrusion of intracellular Mg2+. Meanwhile, silencing SLC41A1 by RNA interference also impaired the elevation of [Ca2+]i, [Mg2+]i efflux and the upregulation of CTGF, FN and α-SMA provoked by Ang II. Furthermore, we found that Ang II-mediated activation of NFATc4 translocation decreased in SLC41A1-siRNA cells. These results support the notion that rapid extrusion of intracellular Mg2+ is mediated by SLC41A1 and provide the evidence that the intracellular free Ca2+ concentration is influenced by extrusion of intracellular Mg2+ which facilitates fibrosis reaction in cardiac fibroblasts.

The microRNA-132/212 family fine-tunes multiple targets in Angiotensin II signalling in cardiac fibroblasts.

MicroRNAs (miRNAs) are emerging as key regulators of cardiovascular development and disease; however, the cardiac miRNA target molecules are not well understood. We and others have described the Angiotensin II (AngII)-induced miR-132/212 family as novel regulators of cardiovascular function including regulation of cardiac hypertrophy, heart failure and blood pressure possibly through AT1R signalling. However, the miR-132/212 targets in the heart remain unknown. To understand the role of these miRNAs in cardiac signalling networks, we undertook comprehensive in silico and in vitro experiments to identify miR-132/212 molecular targets in primary rat cardiac fibroblasts. MiR-132/212 overexpression increased fibroblast cell size and mRNA arrays detected several hundred genes that were differentially expressed, including a wide panel of receptors, signalling molecules and transcription factors. Subsequent comprehensive in silico analysis identified 24 target genes, of which 22 genes were qPCR validated. We identified seven genes involved in AngII signalling pathways. We here report novel insight of an extensive network of molecular pathways that fine-tuned by miR-132/212, suggesting a role for this miRNA family as master signalling switches in cardiac fibroblasts. Our data underscore the potential for miRNA tools to manipulate a large array of molecules and thereby control biological function.

Inhibition of high-mobility group box 1 improves myocardial fibrosis and dysfunction in diabetic cardiomyopathy

BackgroundHigh-mobility group box 1 (HMGB1) is an important mediator of the inflammatory response. Its expression is increased in diabetic cardiomyopathy (DCM), but its role is unclear. We investigated the potential role and mechanism of HMGB1 in diabetes-induced myocardial fibrosis and dysfunction in mice. MethodsIn vivo, type 1 diabetes was induced by streptozotocin (STZ) in mice. HMGB1 expression was knocked down by lentivirus-mediated short-hairpin RNA (shRNA). Cardiac function was assessed by echocardiography. Total collagen deposition was assessed by Masson's trichrome and Picrosirius red staining. HMGB1, collagen I and III, and transforming growth factor β1 (TGF-β1) expression was quantified by immunostaining and western bolt analysis. In vitro, isolated neonatal cardiac fibroblasts were treated with high glucose (HG) or recombinant HMGB1 (rHMGB1). Pharmacologic (neutralizing anti-HMGB1 antibody) or genetic (shRNA-HMGB1) inhibition of HMGB1 was used to investigate the role of HMGB1 in HG-induced functional changes of cardiac fibroblasts. ResultsIn vivo, HMGB1 was diffusely expressed in the myocardium of diabetic mice. HMGB1 silencing ameliorated left ventricular dysfunction and remodeling and decreased collagen deposition in diabetic mice. In vitro, HG induced HMGB1 translocation and secretion in both viable cardiomyocytes and fibroblasts. Administration of rHMGB1 dose-dependently increased the expression of collagens I and III and TGF-β1 in cardiac fibroblasts. HMGB1 inhibition reduced HG-induced collagen production, matrix metalloproteinase (MMP) activities, proliferation, and activated mitogen-activated protein kinase signaling in cardiac fibroblasts. ConclusionsHMGB1 inhibition could alleviate cardiac fibrosis and remodeling in diabetic cardiomyopathy. Inhibition of HMGB1 might have therapeutic potential in the treatment of the disease.

A-kinase anchoring protein-Lbc promotes pro-fibrotic signaling in cardiac fibroblasts

In response to stress or injury the heart undergoes an adverse remodeling process associated with cardiomyocyte hypertrophy and fibrosis. Transformation of cardiac fibroblasts to myofibroblasts is a crucial event initiating the fibrotic process. Cardiac myofibroblasts invade the myocardium and secrete excess amounts of extracellular matrix proteins, which cause myocardial stiffening, cardiac dysfunctions and progression to heart failure. While several studies indicate that the small GTPase RhoA can promote profibrotic responses, the exchange factors that modulate its activity in cardiac fibroblasts are yet to be identified. In the present study, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor (GEF) activity, is critical for activating RhoA and transducing profibrotic signals downstream of type I angiotensin II receptors (AT1Rs) in cardiac fibroblasts. In particular, our results indicate that suppression of AKAP-Lbc expression by infecting adult rat ventricular fibroblasts with lentiviruses encoding AKAP-Lbc specific short hairpin (sh) RNAs strongly reduces the ability of angiotensin II to promote RhoA activation, differentiation of cardiac fibroblasts to myofibroblasts, collagen deposition as well as myofibroblast migration. Interestingly, AT1Rs promote AKAP-Lbc activation via a pathway that requires the α subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as a key Rho-guanine nucleotide exchange factor modulating profibrotic responses in cardiac fibroblasts.

252 SIGNAL MODULATION IN CARDIAC FIBROBLASTS BY THE PLASMA MEMBRANE CALCIUM ATPASE 4 (PMCA4) CONTROLS CARDIAC HYPERTROPY

Cardiac bypertrophy is an important pathological process leading to heart failure (HF). The identification of novel regulators of myocardial hypertrophy is key in understanding the mechanisms of HF and in finding new treatment modalities. PMCA4 is a ubiquitously expressed Ca²⁺ pump that is involved in molecular signalling in the heart. Here we have investigated a novel role of PMCA4 in cardiac fibroblasts and the cross-talk signalling between PMCA4-deficient fibroblasts and cardiomyocytes in controlling myocardial hypertrophy. To study the role of PMCA4 during hypertrophy we subjected PMCA4^−/− mice to transverse aortic constriction (TAC) for 5 weeks. PMCA4^−/− mice exhibited a significantly reduced hypertrophic response compared with wild type (WT) mice (Heart weight/tibia length ratio after TAC, PMCA4^−/−: 6.74+0.33 mg/mm vs WT: 8.34+0.88 mg/mm, P&lt;0.05, n&gt;10). This was accompanied by less fibrosis and lower expression of the hypertrophic marker BNP. However, cardiomyocyte specific knockout of PMCA4 did not produce any protective effect in response to TAC for 5 weeks. We reasoned that the phenotype might be due to PMCA4-mediated signalling in fibroblasts. Microarray analysis revealed a massive upregulation (∼100 fold) of secreted frizzled-related protein 2 (sFRP2) in PMCA4^−/− fibroblasts, which was confirmed by Western blot and real time PCR analyses. sFRP2 is a potent inhibitor of the pro-hypertrophic Wnt/β-catenin signalling pathway. To further test the anti-hypertrophic properties of PMCA4^−/− fibroblasts, we co-cultured WT cardiomyocytes with either PMCA4^−/− or WT fibroblasts. In response to phenylephrine stimulation cardiomyocytes cultured with PMCA4^−/− fibroblasts displayed 88% less hypertrophy than those cultured with WT fibroblasts (P&lt;0.01). Moreover, addition of anti-sFRP2 antibody to the culture medium abolished the anti-hypertrophic effect of PMCA4^−/− fibroblasts, suggesting that the phenotype might be due to the paracrine effect of secreted sFRP2. Mechanistically, we found that PMCA4^−/− fibroblasts showed a significant elevation in NFkB activity, an essential transcription factor regulating sFRP2 expression. Inhibition of NFkB activity using a specific inhibitor Bay11-7085 reduced the expression of sFRP2 in PMCA4^−/− fibroblasts to the level comparable with WT expression. In conclusion, our data provides new evidence that PMCA4-mediated signalling in cardiac fibroblasts plays a key role in controlling cardiac hypertrophy. Cardiac fibroblasts lacking PMCA4 produce higher levels of the protective protein sFRP2 which in turn inhibits the hypertrophic response in the neighbouring cardiomyocytes. Our study also demonstrates the importance of signalling crosstalk between cardiac fibroblasts and cardiomyocytes both <i>in vitro</i> and <i>in vivo</i>.

β3 Integrin in Cardiac Fibroblast Is Critical for Extracellular Matrix Accumulation during Pressure Overload Hypertrophy in Mouse

The adhesion receptor β3 integrin regulates diverse cellular functions in various tissues. As β3 integrin has been implicated in extracellular matrix (ECM) remodeling, we sought to explore the role of β3 integrin in cardiac fibrosis by using wild type (WT) and β3 integrin null (β3−/−) mice for in vivo pressure overload (PO) and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, β3−/− mice upon pressure overload hypertrophy for 4 wk by transverse aortic constriction (TAC) showed a substantially reduced accumulation of interstitial fibronectin and collagen. Moreover, pressure overloaded LV from β3−/− mice exhibited reduced levels of both fibroblast proliferation and fibroblast-specific protein-1 (FSP1) expression in early time points of PO. To test if the observed impairment of ECM accumulation in β3−/− mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and β3−/− mice for adhesion to ECM proteins, cell spreading, proliferation, and migration in response to platelet derived growth factor-BB (PDGF, a growth factor known to promote fibrosis) stimulation. Our results showed that β3−/− cardiac fibroblasts exhibited a significant reduction in cell-matrix adhesion, cell spreading, proliferation and migration. In addition, the activation of PDGF receptor associated tyrosine kinase and non-receptor tyrosine kinase Pyk2, upon PDGF stimulation were impaired in β3−/− cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F) resulted in reduced accumulation of fibronectin. These results indicate that β3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis.

Reduced Endoglin Activity Limits Cardiac Fibrosis and Improves Survival in Heart Failure

Heart failure is a major cause of morbidity and mortality worldwide. The ubiquitously expressed cytokine transforming growth factor-β1 (TGFβ1) promotes cardiac fibrosis, an important component of progressive heart failure. Membrane-associated endoglin is a coreceptor for TGFβ1 signaling and has been studied in vascular remodeling and preeclampsia. We hypothesized that reduced endoglin expression may limit cardiac fibrosis in heart failure. We first report that endoglin expression is increased in the left ventricle of human subjects with heart failure and determined that endoglin is required for TGFβ1 signaling in human cardiac fibroblasts using neutralizing antibodies and an siRNA approach. We further identified that reduced endoglin expression attenuates cardiac fibrosis, preserves left ventricular function, and improves survival in a mouse model of pressure-overload-induced heart failure. Prior studies have shown that the extracellular domain of endoglin can be cleaved and released into the circulation as soluble endoglin, which disrupts TGFβ1 signaling in endothelium. We now demonstrate that soluble endoglin limits TGFβ1 signaling and type I collagen synthesis in cardiac fibroblasts and further show that soluble endoglin treatment attenuates cardiac fibrosis in an in vivo model of heart failure. Our results identify endoglin as a critical component of TGFβ1 signaling in the cardiac fibroblast and show that targeting endoglin attenuates cardiac fibrosis, thereby providing a potentially novel therapeutic approach for individuals with heart failure.

β3 integrin/PDGF receptor synergistic signaling mediates cardiac fibrosis in a mouse model of pressure overload hypertrophy

The precise role of integrins in the process of cardiac fibrosis is not well understood. We hypothesized that upon cardiac hypertrophic stimulation, the synergistic activation of β3 integrin and platelet‐derived growth factor receptor (PDGFR) leading to nonreceptor tyrosine kinase (NTK) signaling in cardiac fibroblasts contributes to fibrosis. In response to pressure‐overload induced by transverse aortic constriction (TAC), β3 integrin knockout (β3 KO) mice exhibited reduced fibrillar collagen, fibronectin, and fibroblast specific protein (FSP1) levels in comparison to wild‐type (WT) mice. Isolated cardiac fibroblasts (Cfb) from β3 KO mice exhibited a reduction in cell adhesion and spreading as well as PDGF‐induced cell proliferation and migration when compared to Cfb from WT mice. Next, we analyzed the phosphorylation state of PDGFR and NTK upon PDGF stimulation in Cfb. Our data indicated a marked reduction in the activation of PDGFR, Pyk2, Src and FAK in β3 KO Cfb when compared to WT Cfb. These data suggest that β3 integrin provides a necessary co‐stimulation for fibrotic signaling in Cfb. Furthermore, our studies in the mouse TAC model where dasatinib, an inhibitor of PDGFR and NTK, was administered via a mini‐osmotic pump, exhibited a preserved ventricular geometry and function and reduced fibrosis upon 4 wk TAC. NTK inhibition with dasatinib also reduced the proliferation, migration and mitogenic signaling of Cfb. These data indicate that integrin‐PDGFR synergism could be a potential target to treat cardiac fibrosis.