Gene expression changes associated with fibronectin-induced cardiac myocyte hypertrophy

Abstract

(FN) is an extracellular matrix protein that binds to integrin receptors and couples cardiac myocytes to the basal lamina. Cardiac FN expression is elevated in models of pressure overload, and FN causes cultured cardiac myocytes to hypertrophy by a mechanism that has not been characterized in detail. In this study, we analyzed the gene expression changes induced by FN in purified rat neonatal ventricular myocytes using the Affymetrix RAE230A microarray, to understand how FN affects gene expression in cardiac myocytes and to separate the effects contributed by cardiac nonmyocytes in vivo. Pathway analysis using z-score statistics and comparison with a mouse model of cardiac hypertrophy revealed several pathways stimulated by FN in cardiac myocytes. In addition to the known cardiac myocyte hypertrophy markers, FN significantly induced metabolic pathways including virtually all of the enzymes of cholesterol biosynthesis, fatty acid biosynthesis, and the mitochondrial electron transport chain. FN also increased the expression of genes coding for ribosomal proteins, translation factors, and the ubiquitin-proteasome pathway. Interestingly, cardiac myocytes plated on FN showed elevated expression of the fibrosis-promoting peptides connective tissue growth factor (CTGF), WNT1 inducible signaling pathway protein 2 (WISP2), and secreted acidic cysteine-rich glycoprotein (SPARC). Our data complement in vivo studies and reveal several novel genes and pathways stimulated by FN, pointing to cardiac myocyte-specific mechanisms that lead to development of the hypertrophic phenotype. extracellular matrix; integrin; ventricular remodeling; signal transduction; gene expression profiling OUTSIDE-IN SIGNALING CHARACTERIZES the cellular effects mediated by interaction of cellular receptors with the extracellular matrix. In neonatal rat ventricular myocytes (NRVM), clustering of integrin receptors induced by fibronectin (FN) (39,40) or RGD peptides (3) elicits a hypertrophic response, characterized by a 1.5-to 3-fold increase in cellular area and corresponding increases in global mRNA and protein synthesis, together with enhanced myofibrillogenesis and sarcomeric assembly. FN also induces increased formation of focal adhesions and costameres, which are sites of contact of extracellular matrix with integrins and associated cytoskeletal proteins associated with the Z-disks of the sarcomere (39). As in other models of hypertrophy, the hypertrophic response is accompanied by changes in gene expression, with increased expression of the natriuretic peptides atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) and fetal isoforms of sarcomeric proteins such as ␤-myosin heavy chain (␤MHC) and ␣-skeletal actin (␣SkA).The pathophysiological relevance of the FN-induced, integrin-mediated pathway is highlighted by the observation of increased deposition of FN in the extracellular matrix in animal models of cardiac hypertrophy and in patients with cardiac failure (5,20,22,29,42,45). The increased synthesis of FN is in part mediated...

Transcriptome of hypertension-induced left ventricular hypertrophy and its regression by antihypertensive therapies

“…27,28 Clustered with Akt and gp130 was fibroblast growth factor-1, an angiogenic growth factor that has earlier been shown in human cardiac hypertrophy 29 and that may reflect that blood vessel recruitment is an important feature of normal tissue growth. 30 An increase in connective tissue growth factor (CTGF) expression was seen in late LVH, similar to findings in other models of cardiac hypertrophy, 31 and its well-characterized profibrotic effect, including its ability to induce cardiac fibroblast proliferation and also extracellular matrix expansion. 32 Hayata et al 33 have shown that cardiac myocytes stimulated with CTGF and its C-terminalregion peptide showed an increase in the cell surface area.…”

Role of the integrin-linked kinase/PINCH1/alpha-parvin complex in cardiac myocyte hypertrophy

“…Ventricular myocytes were isolated from 1-or 2-dayold newborn Sprague-Dawley rats according to an established protocol 26,30 and in compliance with the 'Guiding Principles for the Care and Use of Animals.' In brief, ventricles were digested with trypsin and DNAse I and nonmyocytes were removed by preplating in uncoated tissue culture dishes for 1 h. Nonattached cardiac myocytes were plated in tissue culture dishes that were precoated with 0.5 mg/cm 2 of FN (Sigma, St Louis, MO, USA) or with 100 mg/cm 2 of gelatin for 3-4 h. Cells were usually plated at a density of 500 cells/mm 2 and cultured for 24 h in MEM containing 5% bovine calf serum and 0.1 mM bromodeoxyuridine at 371C in 1% CO 2 .…”

“…26 Briefly, cardiac myocytes were cultured in eight-well slides, fixed in PBS containing 2% paraformaldeyde, permeabilized with 0.1% Triton X-100, and stained with primary and secondary antibodies diluted in PBS containing 0.1% Tween 20 (PBS-T) and 5% normal donkey serum. Pimary antibodies included rabbit anti-atrial natriuretic peptide (ANP, Peninsula Laboratories, Belmont, CA, USA), mouse clone 65.1 anti-ILK, anti-PARVA antibody 3B5, and anti-sarcomeric actinin antibody (Sigma #A7811).…”

“…24 Interestingly, ILK and IPAP1 complex assembly are required for cytoskeletal association of paxillin in epithelial cells 14 and a significant increase in ILK mRNA has been observed in a pressure-overload mouse model of cardiac hypertrophy. 25 Studies from our laboratory 26 and others 27,28 have demonstrated that FN induces cardiac myocyte hypertrophy in neonatal rat ventricular myocytes (NRVM). Moreover, FN-induced hypertrophy depended on intact integrin signaling, as evidenced by inhibition with anti-b1-integrin antibody, [26][27][28] and potentiated hypertrophy induced by the alphaadrenergic agonist PE.…”

“…25 Studies from our laboratory 26 and others 27,28 have demonstrated that FN induces cardiac myocyte hypertrophy in neonatal rat ventricular myocytes (NRVM). Moreover, FN-induced hypertrophy depended on intact integrin signaling, as evidenced by inhibition with anti-b1-integrin antibody, [26][27][28] and potentiated hypertrophy induced by the alphaadrenergic agonist PE. 29 In this study, we examine the hypothesis that FN and PE stimulation regulate the expression and localization of ILK, PARVA and PINCH1 proteins in cardiac myocytes where they may play a role in promoting cellular survival and transducing extracellular signals leading to hypertrophy.…”