Ryan H. Cunnington

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 α‐smooth muscle actin (αSMA), smooth muscle myosin heavy chain (SMemb), extra domain‐A (ED‐A) fibronectin, paxillin, tensin, FGF‐2, and TβRII 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. Developmental Dynamics 239:1573–1584, 2010.

Apoptosis, autophagy and ER stress in mevalonate cascade inhibition-induced cell death of human atrial fibroblasts

3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) are cholesterol-lowering drugs that exert other cellular effects and underlie their beneficial health effects, including those associated with myocardial remodeling. We recently demonstrated that statins induces apoptosis and autophagy in human lung mesenchymal cells. Here, we extend our knowledge showing that statins simultaneously induces activation of the apoptosis, autophagy and the unfolded protein response (UPR) in primary human atrial fibroblasts (hATF). Thus we tested the degree to which coordination exists between signaling from mitochondria, endoplasmic reticulum and lysosomes during response to simvastatin exposure. Pharmacologic blockade of the activation of ER-dependent cysteine-dependent aspartate-directed protease (caspase)-4 and lysosomal cathepsin-B and -L significantly decreased simvastatin-induced cell death. Simvastatin altered total abundance and the mitochondrial fraction of proapoptotic and antiapoptotic proteins, while c-Jun N-terminal kinase/stress-activated protein kinase mediated effects on B-cell lymphoma 2 expression. Chemical inhibition of autophagy flux with bafilomycin-A1 augmented simvastatin-induced caspase activation, UPR and cell death. In mouse embryonic fibroblasts that are deficient in autophagy protein 5 and refractory to autophagy induction, caspase-7 and UPR were hyper-induced upon treatment with simvastatin. These data demonstrate that mevalonate cascade inhibition-induced death of hATF manifests from a complex mechanism involving co-regulation of apoptosis, autophagy and UPR. Furthermore, autophagy has a crucial role in determining the extent of ER stress, UPR and permissiveness of hATF to cell death induced by statins.

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts.

Transforming growth factor-β1 (TGF-β1) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β1 may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β1-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β1 to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β1 promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β1 in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β1-induced autophagy is required for the fibrogenic response in hATMyofbs.

The participation of the Na+–Ca2+ exchanger in primary cardiac myofibroblast migration, contraction, and proliferation

Cardiac ventricular myofibroblast motility, proliferation, and contraction contribute to post‐myocardial infarct wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The Na+–Ca2+ exchanger (NCX1) is involved in altered calcium handling in cardiac myocytes during cardiac remodeling associated with heart failure, however, its role in cardiac myofibroblast cell function is unexplored. In this study we investigated the involvement of NCX1 as well as the role of non‐selective‐cation channels (NSCC) in cardiac myofibroblast cell function in vitro. Immunofluorescence and Western blots revealed that P1 cells upregulate α‐smooth muscle actin (αSMA) and embryonic smooth muscle myosin heavy chain (SMemb) expression. NCX1 mRNA and proteins as well as Cav1.2a protein are also expressed in P1 myofibroblasts. Myofibroblast motility in the presence of 50 ng/ml PDGF‐BB was blocked with AG1296. Myofibroblast motility, contraction, and proliferation were sensitive to KB‐R7943, a specific NCX1 reverse‐mode inhibitor. In contrast, only proliferation and contraction, but not motility were sensitive to nifedipine, while gadolinium (NSCC blocker) was only associated with decreased motility. ML‐7 treatment was associated with inhibition of the chemotactic response and contraction. Thus cardiac myofibroblast chemotaxis, contraction, and proliferation were sensitive to different pharmacologic treatments suggesting that regulation of transplasmalemmal calcium movements may be important in growth factor receptor‐mediated processes. NCX1 may represent an important moiety in suppression of myofibroblast functions. J. Cell. Physiol. 213: 540–551, 2007.

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-β₁ (TGF-β₁). The c-Ski proto-oncoprotein has been shown to modify TGF-β₁ 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-β₁ (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-β₁ 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.

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-β 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.

The Ski-Zeb2-Meox2 pathway provides a novel mechanism for regulation of the cardiac myofibroblast phenotype

ABSTRACT Cardiac fibrosis is linked to fibroblast-to-myofibroblast phenoconversion and proliferation but the mechanisms underlying this are poorly understood. Ski is a negative regulator of TGF-&bgr;–Smad signaling in myofibroblasts, and might redirect the myofibroblast phenotype back to fibroblasts. Meox2 could alter TGF-&bgr;-mediated cellular processes and is repressed by Zeb2. Here, we investigated whether Ski diminishes the myofibroblast phenotype by de-repressing Meox2 expression and function through repression of Zeb2 expression. We show that expression of Meox1 and Meox2 mRNA and Meox2 protein is reduced during phenoconversion of fibroblasts to myofibroblasts. Overexpression of Meox2 shifts the myofibroblasts into fibroblasts, whereas the Meox2 DNA-binding mutant has no effect on myofibroblast phenotype. Overexpression of Ski partially restores Meox2 mRNA expression levels to those in cardiac fibroblasts. Expression of Zeb2 increased during phenoconversion and Ski overexpression reduces Zeb2 expression in first-passage myofibroblasts. Furthermore, expression of Meox2 is decreased in scar following myocardial infarction, whereas Zeb2 protein expression increases in the infarct scar. Thus Ski modulates the cardiac myofibroblast phenotype and function through suppression of Zeb2 by upregulating the expression of Meox2. This cascade might regulate cardiac myofibroblast phenotype and presents therapeutic options for treatment of cardiac fibrosis.

c-Ski, Smurf2, and Arkadia as regulators of TGF-β signaling: new targets for managing myofibroblast function and cardiac fibrosisThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.

Recent studies demonstrate the critical role of the extracellular matrix in the organization of parenchymal cells in the heart. Thus, an understanding of the modes of regulation of matrix production by cardiac myofibroblasts is essential. Transforming growth factor beta (TGF-beta) signaling is transduced through the canonical Smad pathway, and the involvement of this pathway in matrix synthesis and other processes requires precise control. Inhibition of Smad signaling may be achieved at the receptor level through the targeting of the TGF-beta type I receptors with an inhibitory Smad7/Smurf2 complex, or at the transcriptional level through c-Ski/receptor-Smad/co-mediator Smad4 interactions. Conversely, Arkadia protein intensifies TGF-beta-induced effects by marking c-Ski and inhibitory Smad7 for destruction. The study of these TGF-beta mediators is essential for future treatment of fibrotic disease, and this review highlights recent relevant findings that may impact our understanding of cardiac fibrosis.

Mast Cells and Cardiac Fibroblasts: Accomplices in Elevation of Collagen Synthesis in Modulation of Fibroblast Phenotype

See related article, pp 264–270 The term “fibroblast” designates a highly heterogenous group of cells that exhibit distinct differentiated phenotypes in different organs.1 The study of fibroblast and myofibroblast biology in specific organs is an important but relatively understudied area, especially in the heart. Recent novel data indicate that ventricular fibroblast activation and cardiac fibrosis are primary events in ventricular remodeling rather than secondary to cardiomyocyte injury.2 In larger mammalian species, including humans, cardiac fibroblasts represent the most numerous nonmyocytes in the myocardium. These cells function to synthesize and organize collagens, fibronectins, and other interstitial components and, thus, maintain the integrity of the cardiac extracellular matrix (matrix). Matrix remodeling can manifest as interstitial fibrosis of an otherwise normal myocardium. This remodeling may occur with the onset of hypertension or as the progressive evolution of the structure of the infarct scar. Remodeling of the matrix occurs later in the noninfarcted myocardium, in the etiology of heart failure after myocardial infarction. The dogma that cardiac fibrosis is merely a secondary disease modifier after cardiomyocyte damage is losing ground to the idea that fibrosis is a primary disease prima facie. Therefore, the need to identify and characterize the specific signals that might trigger the phenoconversion of relatively quiescent fibroblasts to myofibroblasts takes on new importance. In normal heart tissue, matrix protein secretion and deposition are carried out exclusively by cardiac fibroblasts with relatively low turnover of proteins. Conversely, contractile and hypersynthetic myofibroblasts are the relevant phenotypic variants in wound healing or in hypertrophied and …

Regulatory Role of TGF-β in Cardiac Myofibroblast Function and Post-MI Cardiac Fibrosis: Key Roles of Smad7 and c-Ski

The molecular pathways that couple increased hemodynamic load to cardiac hypertrophy, cardiac fibrosis, and heart failure are incompletely understood. Cardiac fibrosis is recognized as a major disease modifier and as such is important in the pathogenesis of heart failure of most etiologies. This review is focused on R-Smad signaling in cardiac myofibroblasts and their role in remodeling the extracellular matrix of the failing myocardium after myocardial infarction (MI). As major mediators of TGF-β1 signaling in cardiac fibroblasts and myofibroblasts as well as myocytic cells, Smad proteins are emerging as an important postreceptor class in post-MI heart failure. How cytosolic c-Ski and C184M proteins influence R-Smads (and possibly Smad7 itself) in cardiac myofibroblasts, and how c-Ski expression influences cardiac myofibroblast function are largely unknown. We suggest that decreased Smad7 expression and altered intracellular c-Ski expression and/or distribution may contribute to chronic imbalance of R-Smad activation in classic TGF-β1 signaling in the context of cardiac myofibroblast function. Thus, reduced and insufficient expression and activation of c-Ski and Smad7 may contribute to abnormal stimulation of collagen synthesis by these cells. Exploitation of Smad7 and c-Ski in the modulation of cardiac myofibroblast function may provide the experimental basis for the development of highly specific drugs for treating heart failure with attendant cardiac fibrosis. Future studies to identify and characterize Smad-associated factors responsible for induction of pathological cardiac hypertrophy are warranted.