Primary Cardiac Myofibroblasts Research Articles

Ski drives an acute increase in MMP-9 gene expression andrelease in primary cardiac myofibroblasts.

Many etiologies of heart disease are characterized by expansion and remodeling of the cardiac extracellular matrix (ECM or matrix) which results in cardiac fibrosis. Cardiac fibrosis is mediated in cardiac fibroblasts by TGF‐β 1/R‐Smad2/3 signaling. Matrix component proteins are synthesized by activated resident cardiac fibroblasts known as myofibroblasts (MFB). These events are causal to heart failure with diastolic dysfunction and reduced cardiac filling. We have shown that exogenous Ski, a TGF‐β 1/Smad repressor, localizes in the cellular nucleus and deactivates cardiac myofibroblasts. This deactivation is associated with reduction of myofibroblast marker protein expression in vitro, including alpha smooth muscle actin (α‐SMA) and extracellular domain‐A (ED‐A) fibronectin. We hypothesize that Ski also acutely regulates MMP expression in cardiac MFB. While acute Ski overexpression in cardiac MFB in vitro was not associated with any change in intracellular MMP‐9 protein expression versus LacZ‐treated controls,exogenous Ski caused elevated MMP‐9 mRNA expression and increased MMP‐9 protein secretion versus controls. Zymographic analysis revealed increased MMP‐9‐specific gelatinase activity in myofibroblasts overexpressing Ski versus controls. Moreover, Ski expression was attended by reduced paxillin and focal adhesion kinase phosphorylation (FAK ‐ Tyr 397) versus controls. As myofibroblasts are hypersecretory and less motile relative to fibroblasts, Ski's reduction of paxillin and FAK expression may reflect the relative deactivation of myofibroblasts. Thus, in addition to its known antifibrotic effects, Ski overexpression elevates expression and extracellular secretion/release of MMP‐9 and thus may facilitate internal cytoskeletal remodeling as well as extracellular ECM components. Further, as acute TGF‐β 1 treatment of primary cardiac MFB is known to cause rapid translocation of Ski to the nucleus, our data support an autoregulatory role for Ski in mediating cardiac ECM accumulation.

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 TGFβ1/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.

TGFβ1 regulates Scleraxis expression in primary cardiac myofibroblasts by a Smad-independent mechanism.

In cardiac wound healing following myocardial infarction (MI), relatively inactive resident cardiac fibroblasts phenoconvert to hypersynthetic/secretory myofibroblasts that produce large quantities of extracellular matrix (ECM) and fibrillar collagen proteins. Our laboratory and others have identified TGFβ1 as being a persistent stimulus in the chronic and inappropriate wound healing phase that is marked by hypertrophic scarring and eventual stiffening of the entire myocardium, ultimately leading to the pathogenesis of heart failure following MI. Ski is a potent negative regulator of TGFβ/Smad signaling with known antifibrotic effects. Conversely, Scleraxis is a potent profibrotic basic helix-loop-helix transcription factor that stimulates fibrillar collagen expression. We hypothesize that TGFβ1 induces Scleraxis expression by a novel Smad-independent pathway. Our data support the hypothesis that Scleraxis expression is induced by TGFβ1 through a Smad-independent pathway in the cardiac myofibroblast. Specifically, we demonstrate that TGFβ1 stimulates p42/44 (Erk1/2) kinases, which leads to increased Scleraxis expression. Inhibition of MEK1/2 using U0126 led to a sequential temporal reduction of phospho-p42/44 and subsequent Scleraxis expression. We also found that adenoviral Ski expression in primary myofibroblasts caused a significant repression of endogenous Scleraxis expression at both the mRNA and protein levels. Thus we have identified a novel TGFβ1-driven, Smad-independent, signaling cascade that may play an important role in regulating the fibrotic response in activated cardiac myofibroblasts following cardiac injury.

Angiotensin-converting enzyme 2 is subject to post-transcriptional regulation by miR-421

ACE2 (angiotensin converting enzyme 2) plays a critical role in the local tissue RAS (renin-angiotensin system) by hydrolysing the potent hypertensive and mitogenic peptide AngII (angiotensin II). Changes in the levels of ACE2 have been observed in a number of pathologies, including cardiovascular disease, but little is known of the mechanisms regulating its expression. In the present study, therefore, the potential role of miRNAs in the regulation of ACE2 expression in primary human cardiac myofibroblasts was examined. Putative miRNA-binding sites were identified in the 3'-UTR of the ACE2 transcript using online prediction algorithms. Two of these, miR-200b and miR-421, were selected for further analysis. A reporter system using the 3'-UTR of ACE2 fused to the coding region of firefly luciferase was used to determine the functionality of the identified binding sites invitro. This identified miR-421, but not miR-200b, as a potential regulator of ACE2. The ability of miR-421, an miRNA implicated in the development of thrombosis, to down-regulate ACE2 expression was subsequently confirmed by Western blot analysis of both primary cardiac myofibroblasts and transformed cells transfected with a synthetic miR-421 precursor. Real-time PCR analysis of miR-421 revealed widespread expression in human tissues. miR-421 levels in cardiac myofibroblasts showed significant inter-patient variability, in keeping with the variability of ACE2 expression we have observed previously. In conclusion, the present study is the first to demonstrate that ACE2 may be subject to post-transcriptional regulation and reveals a novel potential therapeutic target, miR-421, which could be exploited to modulate ACE2 expression in disease.

Expression of Ski induces apoptosis and represses autophagy in cardiac myofibroblasts (868.8)

Following myocardial infarction (MI), relatively quiestent cardiac fibroblasts undergo phenoconversion to active, hyper‐synthetic myofibroblasts. During the late stages of wound healing, myofibroblasts are removed from the infracted region through apoptosis thus decreasing scar cellularity. Apoptosis and autophagy regulate cell fate. The role of apoptosis within the remodeling heart has been well described however the role that autophagy plays following an MI is relatively unknown. Ski is a negative regulator of TGF‐β signaling and has been implicated to play a role in the post‐MI heart. Herein we investigated the regulatory role that Ski has on apoptosis and autophagy using first passage primary rat cardiac myofibroblasts. Using an adenoviral approach for gene delivery, both MTT and Live/Dead viability/cytoxicity assays demonstrated a reduction in the number of viable cells and an increase in the number of dying cells due to the over‐expression of Ski. Additionally, over‐expression of Ski led to distinct morphological changes characteristic of apoptosis. These findings were confirmed via Western blot analysis, and caspase GLO analysis showing induction of executioner caspases‐3 and ‐7, as well as the mitochondrial regulator Bax. Furthermore, evidence of caspase‐9 cleavage, but not caspase‐8, indicated that this was primarily an intrinsic apoptotic response. Markers for autophagosome formation including LC3‐B and ATG‐7 were found to be significantly reduced following Ski over‐expression. In summary, we found that over‐expression of Ski in primary cardiac myofibroblasts leads to an induction of the intrinsic apoptotic pathway and reduction in autophagy leading to cell death.Grant Funding Source: Canadian Institutes of Health Research

Identification of novel scleraxis gene targets in cardiac myofibroblasts

Cardiac fibrosis is a significant clinical problem yet lacks effective therapies. We have shown that the transcription factor scleraxis is a key regulator of collagen 1α2 gene expression in cardiac myofibroblasts. We now identify novel scleraxis gene targets via gain‐/loss‐of‐function. Scleraxis over‐expression in primary cardiac myofibroblasts decreased the expression of matrix remodeling enzymes matrix metalloproteinases 9 and 11 and tissue inhibitor of metalloproteinases 3. Expression of the proteoglycans versican and decorin were decreased following scleraxis overexpression, yet we observed no change in decorin and a loss of versican in scleraxis null hearts. The expression of thrombospondin 4 and the myofibroblast marker α‐smooth muscle actin (αSMA) was increased by scleraxis overexpression and strongly down‐regulated in scleraxis null hearts, and may thus represent direct scleraxis gene targets. Our results show that scleraxis exerts wide‐ranging effects on extracellular matrix‐related gene expression, with the effect on αSMA expression suggesting that scleraxis may contribute to the fibroblast‐to‐myofibroblast transition that accompanies cardiac fibrosis, further supporting our general hypothesis that scleraxis represents a novel therapeutic target for this disease.Supported by the Canadian Institutes of Health Research & the St. Boniface Hospital Research Foundation.

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, 0–72h) 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.

Transfat‐mediated apoptosis is regulated by autophagy in primary cardiac myofibroblasts

Dietary trans fatty acids (TFA) are linked to cardiovascular disease which has led to recommendations to remove TFAs from diet. We investigated the effects of two common fatty acids ‐ vaccenic acid (VA ‐ found in dairy products), and elaidic acid (EA ‐ found in hydrogenated vegetable oils) on cell death in primary rat ventricular myofibroblasts (rVF). VA‐ and EA‐treated cells exhibited apoptotic cell death in rVF, as reflected in MTT and FACS assays. A significant reduction in mitochondrial membrane potential and an increase in the Bax/Bcl2 ratio and Bax translocation to the mitochondria was found suggesting apoptosis in the presence of TFA. Elevated cleaved caspase‐9 and caspase‐3 (without caspase‐8), shows that EA and VA treatments induce caspase‐dependent apoptosis. Transmission electron microscopy reveals autophagosome formation and lysosome activation in VA and EA treated rVF. Induction of autophagy markers including LC3‐B lipidation, increased Atg5‐12 formation and increased beclin‐1 were observed confirming autophagy activation by EA and VA. Inhibition of ATG3 and ATG5 significantly inhibited cytotoxic effects by preventing caspase‐3 and caspase‐7 activation in EA and VA treated rVF. In conclusion we show that TFAs induce apoptosis and autophagy in rVF and that TFA‐induced autophagy is required for this proapoptotic effect. Supported by CIHR and DFC.

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.