SKI activates the Hippo pathway via LIMD1 to inhibit cardiac fibroblast activation.

We have previously shown that overexpression of SKI, an endogenous TGF-ß1 repressor, deactivates the pro-fibrotic myofibroblast phenotype in the heart. We now show that SKI also functions independently of SMAD/TGF-ß signaling, by activating the Hippo tumor-suppressor pathway and inhibiting the Transcriptional co-Activator with PDZ-binding motif (TAZ or WWTR1). The mechanism(s) by which SKI targets TAZ to inhibit cardiac fibroblast activation and fibrogenesis remain undefined. A rat model of post-myocardial infarction was used to examine the expression of TAZ during acute fibrogenesis and chronic heart failure. Results were then corroborated with primary rat cardiac fibroblast cell culture performed both on plastic and on inert elastic substrates, along with the use of siRNA and adenoviral expression vectors for active forms of SKI, YAP, and TAZ. Gene expression was examined by qPCR and luciferase assays, while protein expression was examined by immunoblotting and fluorescence microscopy. Cell phenotype was further assessed by functional assays. Finally, to elucidate SKI's effects on Hippo signaling, the SKI and TAZ interactomes were captured in human cardiac fibroblasts using BioID2 and mass spectrometry. Potential interactors were investigated in vitro to reveal novel mechanisms of action for SKI. In vitro assays on elastic substrates revealed the ability of TAZ to overcome environmental stimuli and induce the activation of hypersynthetic cardiac myofibroblasts. Further cell-based assays demonstrated that SKI causes specific proteasomal degradation of TAZ, but not YAP, and shifts actin cytoskeleton dynamics to inhibit myofibroblast activation. These findings were supported by identifying the bi-phasic expression of TAZ in vivo during post-MI remodeling and fibrosis. BioID2-based interactomics in human cardiac fibroblasts suggest that SKI interacts with actin-modifying proteins and with LIM Domain-containing protein 1 (LIMD1), a negative regulator of Hippo signaling. Furthermore, we found that LATS2 interacts with TAZ, whereas LATS1 does not, and that LATS2 knockdown prevented TAZ downregulation with SKI overexpression. Our findings indicate that SKI's capacity to regulate cardiac fibroblast activation is mediated, in part, by Hippo signaling. We postulate that the interaction between SKI and TAZ in cardiac fibroblasts is arbitrated by LIMD1, an important intermediary in focal adhesion-associated signaling pathways. This study contributes to the understanding of the unique physiology of cardiac fibroblasts, and of the relationship between SKI expression and cell phenotype.

Novel factors that activate and deactivate cardiac fibroblasts: A new perspective for treatment of cardiac fibrosis.

Heart disease with attendant cardiac fibrosis kills more patients in developed countries than any other disease, including cancer. We highlight the recent literature on factors that activate and also deactivate cardiac fibroblasts. Activation of cardiac fibroblasts results in myofibroblasts phenotype which incorporates aSMA to stress fibres, express ED-A fibronectin, elevated PDGFRα and are hypersecretory ECM components. These cells facilitate both acute wound healing (infarct site) and chronic cardiac fibrosis. Quiescent fibroblasts are associated with normal myocardial tissue and provide relatively slow turnover of the ECM. Deactivation of activated myofibroblasts is a much less studied phenomenon. In this context, SKI is a known negative regulator of TGFb1 /Smad signalling, and thus may share functional similarity to PPARγ activation. The discovery of SKI's potent anti-fibrotic role, and its ability to deactivate and/or myofibroblasts is featured and contrasted with PPARγ. While myofibroblasts are typically recruited from pools of potential precursor cells in a variety of organs, the importance of activation of resident cardiac fibroblasts has been recently emphasised. Myofibroblasts deposit ECM components at an elevated rate and contribute to both systolic and diastolic dysfunction with attendant cardiac fibrosis. A major knowledge gap exists as to specific proteins that may signal for fibroblast deactivation. As SKI may be a functionally pluripotent protein, we suggest that it serves as a scaffold to proteins other than R-Smads and associated Smad signal proteins, and thus its anti-fibrotic effects may extend beyond binding R-Smads. While cardiac fibrosis is causal to heart failure, the treatment of cardiac fibrosis is hampered by the lack of availability of effective pharmacological anti-fibrotic agents. The current review will provide an overview of work highlighting novel factors which cause fibroblast activation and deactivation to underscore putative therapeutic avenues for improving disease outcomes in cardiac patients with fibrosed hearts.

Periostin Reexpression in Heart Disease Contributes to Cardiac Interstitial Remodeling by Supporting the Cardiac Myofibroblast Phenotype.

Cardiac muscle (the myocardium) is a unique arrangement of atria and ventricles that are spatially and electrically separated by a fibrous border. The spirally-arranged myocytes in both left and right ventricles are tethered by the component molecules of the cardiac extracellular matrix (ECM), including fibrillar collagen types I and III. Loss of normal arrangement of the ECM with either too little (as is observed in acute myocardial infarction) or too much (cardiac fibrosis in chronic post-myocardial infarction) is the primary contributor to cardiac dysfunction and heart failure. Matricellular proteins exist as non-structural signaling moieties in the ECM, and in the context of cardiac hypertrophy and heart failure, secreted 90 kDa periostin protein has attracted intense scrutiny during the past decade. Secreted periostin is now recognized for its important role in ECM development and maturation, as well as cellular adhesion. The novel mechanisms of periostin function include its role as a mediator of cell-to-matrix signaling, cell survival, and epithelial-mesenchymal transition (EMT). A number of recent studies have examined the hypothesis that periostin is a major contributor to ECM remodeling in the heart, and a number of very recent studies underscore its important role. This review examines recent developments in the mechanisms of periostin function in the normal heart and vasculature, and discusses recent advances which underpin its putative role in the development of cardiovascular disease. Periostin expression is very low at baseline in healthy tissues, but is re-expressed in damaged heart and in vessel walls after injury, in activated cardiac myofibroblasts and vascular smooth muscle cells, respectively. For this reason, periostin may be exploited for investigation of mechanisms of cardiac fibrosis , and we speculate that data generated from studies utilizing this approach may shed light on the timing for application of periostin-specific therapies to quell cardiac fibrosis and associated cardiac dysfunction.

The Functional Role of Zinc Finger E Box-Binding Homeobox 2 (Zeb2) in Promoting Cardiac Fibroblast Activation.

Following cardiac injury, fibroblasts are activated and are termed as myofibroblasts, and these cells are key players in extracellular matrix (ECM) remodeling and fibrosis, itself a primary contributor to heart failure. Nutraceuticals have been shown to blunt cardiac fibrosis in both in-vitro and in-vivo studies. However, nutraceuticals have had conflicting results in clinical trials, and there are no effective therapies currently available to specifically target cardiac fibrosis. We have previously shown that expression of the zinc finger E box-binding homeobox 2 (Zeb2) transcription factor increases as fibroblasts are activated. We now show that Zeb2 plays a critical role in fibroblast activation. Zeb2 overexpression in primary rat cardiac fibroblasts is associated with significantly increased expression of embryonic smooth muscle myosin heavy chain (SMemb), ED-A fibronectin and α-smooth muscle actin (α-SMA). We found that Zeb2 was highly expressed in activated myofibroblast nuclei but not in the nuclei of inactive fibroblasts. Moreover, ectopic Zeb2 expression in myofibroblasts resulted in a significantly less migratory phenotype with elevated contractility, which are characteristics of mature myofibroblasts. Knockdown of Zeb2 with siRNA in primary myofibroblasts did not alter the expression of myofibroblast markers, which may indicate that Zeb2 is functionally redundant with other profibrotic transcription factors. These findings add to our understanding of the contribution of Zeb2 to the mechanisms controlling cardiac fibroblast activation.

Chronic expression of Ski induces apoptosis and represses autophagy in cardiac myofibroblasts.

Inappropriate cardiac interstitial remodeling is mediated by activated phenoconverted myofibroblasts. The synthesis of matrix proteins by these cells is triggered by both chemical and mechanical stimuli. Ski is a repressor of TGFß1/Smad signaling and has been described as possessing anti-fibrotic properties within the myocardium. We hypothesized that overexpression of Ski in myofibroblasts will induce an apoptotic response, which may either be supported or opposed by autophagic flux. We used primary myofibroblasts (activated fibroblasts) which were sourced from whole heart preparations that were only passaged once. We found that overexpression of Ski results in distinct morphological and biochemical changes within primary cardiac myofibroblasts associated with apoptosis. Ski treatment was associated with the expression of pro-apoptotic factors such as Bax, caspase-7, and -9. Our results indicate that Ski triggers a pro-death mechanism in primary rat cardiac myofibroblasts that is mediated through the intrinsic apoptotic pathway. Myofibroblast survival is prolonged by an autophagic response, as the dataset indicate that apoptosis is hastened when autophagy is inhibited. We suggest that the apoptotic death response of myofibroblasts is working in parallel with the previously observed anti-fibrotic properties of Ski within this cell type. As myofibroblasts are the sole mediators of matrix expansion in heart failure, we suggest that Ski, or a putative Ski-mimetic, may induce graded apoptosis in myofibroblasts within the failing heart and may be a novel therapeutic approach towards controlling cardiac fibrosis. Future studies are needed to examine the potential effects of Ski overexpression on other cell types in the heart.

Autophagy regulates trans fatty acid-mediated apoptosis in primary cardiac myofibroblasts.

Trans fats are not a homogeneous group of molecules and less is known about the cellular effects of individual members of the group. Vaccenic acid (VA) and elaidic acid (EA) are the predominant trans monoenes in ruminant fats and vegetable oil, respectively. Here, we investigated the mechanism of cell death induced by VA and EA on primary rat ventricular myofibroblasts (rVF). The MTT assay demonstrated that both VA and EA (200µM, 0-72 h) reduced cell viability in rVF (P<0.001). The FACS assay confirmed that both VA and EA induced apoptosis in rVF, and this was concomitant with elevation in cleaved caspase-9, -3 and -7, but not caspase-8. VA and EA decreased the expression ratio of Bcl2:Bax, induced Bax translocation to mitochondria and decrease in mitochondrial membrane potential (Δψ). BAX and BAX/BAK silencing in mouse embryonic fibroblasts (MEF) inhibited VA and EA-induced cell death compared to the corresponding wild type cells. Transmission electron microscopy revealed that VA and EA also induced macroautophagosome formation in rVF, and immunoblot analysis confirmed the induction of several autophagy markers: LC3-ß lipidation, Atg5-12 accumulation, and increased beclin-1. Finally, deletion of autophagy genes, ATG3 and ATG5 significantly inhibited VA and EA-induced cell death (P<0.001). Our findings show for the first time that trans fat acid (TFA) induces simultaneous apoptosis and autophagy in rVF. Furthermore, TFA-induced autophagy is required for this pro-apoptotic effect. Further studies to address the effect of TFA on the heart may reveal significant translational value for prevention of TFA-linked heart disease.

Antifibrotic properties of c-Ski and its regulation of cardiac myofibroblast phenotype and contractility.

Cardiac myofibroblasts are key players in chronic remodeling of the cardiac extracellular matrix, which is mediated in part by elevated transforming growth factor-ß1 (TGF-ß1). The c-Ski proto-oncoprotein has been shown to modify TGF-ß1 post-receptor signaling through receptor-activated Smads (R-Smads); however, little is known about how c-Ski regulates fibroblast phenotype and function. We sought to elucidate the function of c-Ski in primary cardiac myofibroblasts using a c-Ski overexpression system. Cardiac myofibroblasts expressed three forms of c-Ski with the predominant band at 105 kDa, and adenoviral c-Ski treatment resulted in overexpression of 95-kDa c-Ski in cellular nuclei. Exogenous c-Ski led to significant inhibition of type I collagen secretion and myofibroblast contractility using two-dimensional semifloating gel contraction assay in both basal and with TGF-ß1 (10 ng/ml for 24 h) stimulation. Overexpressed c-Ski did not inhibit nuclear translocation of phosphorylated R-Smad2, despite their binding, as demonstrated by immunoprecipitation. Acute treatment of primary myofibroblasts with TGF-ß1 in vitro revealed a marked nuclear shuttling of c-Ski at 24 and 48 h following stimulation. Remarkably, overexpression of c-Ski led to a stepwise reduction of the myofibroblast marker α-smooth muscle actin with increasing multiplicity of infection, and these results indicate that 95-kDa c-Ski overexpression may effect a loss of the myofibroblastic phenotype. Furthermore, adenovirus (Ad) for hemagglutinin-tagged c-Ski infection led to a reduction in the number of myofibroblasts versus Ad-LacZ-infected and uninfected controls, due to induction of apoptosis. Finally, we observed a significant increase in 105-kDa c-Ski in the cytosolic fraction of cells of the infarct scar and adjacent remnant myocardium vs. noninfarcted controls.

Role of myosin light chain kinase in cardiotrophin-1-induced cardiac myofibroblast cell migration.

Chemotactic movement of myofibroblasts is recognized as a common means for their sequestration to the site of tissue injury. Following myocardial infarction (MI), recruitment of cardiac myofibroblasts to the infarct scar is a critical step in wound healing. Contractile myofibroblasts express embryonic smooth muscle myosin, α-smooth muscle actin, as well as collagens I and III. We examined the effects of cardiotrophin-1 (CT-1) in the induction of primary rat ventricular myofibroblast motility. Changes in membrane potential (E(m)) and Ca(2+) entry were studied to reveal the mechanisms for induction of myofibroblast migration. CT-1-induced cardiac myofibroblast cell migration, which was attenuated through the inhibition of JAK2 (25 µM AG490), and myosin light chain kinase (20 µM ML-7). Inhibition of K(+) channels (1 mM tetraethylammonium or 100 µM 4-aminopyridine) and nonselective cation channels by 10 µM gadolinium (Gd(3+)) significantly reduced migration in the presence of CT-1. CT-1 treatment caused a significant increase in myosin light chain phosphorylation, which could be inhibited by incubation in Ca(2+)-free conditions or by application of AG490, ML-7, and W7 (100 µM; calmodulin inhibitor). Monitoring myofibroblast membrane potential with potentiometric fluorescent DiBAC(4)(3) dye revealed a biphasic response to CT-1 consisting of an initial depolarization followed by hyperpolarization. Increased intracellular Ca(2+), as assessed by fluo 3, occurred immediately after membrane depolarization and attenuated at the time of maximal hyperpolarization. CT-1 exerts chemotactic effects via multiple parallel signaling modalities in ventricular myofibroblasts, including changes in membrane potential, alterations in intracellular calcium, and activation of a number of intracellular signaling pathways. Further study is warranted to determine the precise role of K(+) currents in this process.

Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro: expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts.

In fibrosing hearts, myofibroblasts are associated with cardiac extracellular matrix remodeling. Expression of key genes in the transition of cardiac fibroblast to myofibroblast phenotype in post-myocardial infarction heart and in vitro has not been well addressed. Contractile, focal adhesion-associated, receptor proteins, fibroblast growth factor-2 (FGF-2) expression, and motility were compared to assess phenotype in adult and neonatal rat cardiac fibroblasts and myofibroblasts. Neonatal and adult fibroblasts undergo phenotypic transition to myofibroblastic cells, marked by increased alpha-smooth muscle actin (alphaSMA), smooth muscle myosin heavy chain (SMemb), extra domain-A (ED-A) fibronectin, paxillin, tensin, FGF-2, and TbetaRII receptor. Elevated ED-A fibronectin confirmed fibroblast to supermature myofibroblastic phenotype transition. Presence of myofibroblasts in vivo was noted in sections of healed infarct scar after myocardial infarction, and their expression is similar to that in culture. Thus, cultured neonatal and adult cardiac fibroblasts transition to myofibroblasts in vitro and share expression profiles of cardiac myofibroblasts in vivo. Reduced motility with in vitro passage reflects enhanced production of focal adhesions.

Soft Substrate Culture to Mechanically Control Cardiac Myofibroblast Activation.

Two-dimensional cell culture is the primary method employed for proof-of-concept studies in most molecular biology labs. While immortalized cell lines are convenient and easy to maintain for extended periods in vitro, their inability to accurately represent genuine cell physiology-or pathophysiology-presents a challenge for drug discovery, as most results are not viable for the transition to clinical trial. The use of primary cells is a more biologically relevant approach to this issue; however, simulating in vitro what is observed in vivo is exigent at best. Primary cardiac fibroblasts are particularly difficult to maintain in a quiescent state, due to their innate phenotypic plasticity, and sensitivity to mechanical and biochemical stimulus. As conventional cell culture methods do not consider these factors, here we describe a method that limits environmental input (i.e., mechanical, nutritional, hormonal) to extend the physiological cardiac fibroblast phenotype in vitro.