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 a-smooth muscle actin (aSMA), smooth muscle myosin heavy chain (SMemb), extra domain-A (ED-A) fibronectin, paxillin, tensin, FGF-2, and TbRII 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,

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.

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.

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.

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Elevated collagen deposition and increased tissue stiffness is the primary contributor to cardiac dysfunction following MI. Ski acts as a cellular brake to modulate phenoconversion of cardiac fibroblasts to myofibroblasts. Ski is associated with reduced collagen and removal of myofibroblasts from damaged heart tissues. Conversely, Scleraxis accelerates expression of collagens in cardiac myofibroblasts. We suggest that a balance exists between pro‐ and anti‐fibrotic pathways in the healthy heart and that Ski and Scleraxis are key mediators.Determine the role that Ski has on Scleraxis expression and the deposition of collagens by cardiac myofibroblasts during cardiac fibrosis.Cardiac fibroblasts were isolated from rat hearts and P1 cardiac myofibroblasts were infected at varying MOI's (10, 25, 50) with sh‐Ski or sh‐EGFP (50) control virus for 6 days. Cells were harvested for cell phenotyping and analysis of fibrillar collagens.A progressive increase in Scleraxis protein expression was noted with increasing sh‐Ski MOI from 10 to 50. This corresponded with a significant increase in mature collagen protein expression suggesting that Ski regulates Scleraxis ability to induce collagen synthesis by cardiac myofibroblasts in vitro.Ski inhibits Scleraxis function eg, to stimulate expression of collagens in cardiac myofibroblasts indicating the existence of a feedback loop between Ski and Scleraxis.

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.