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. 2011 Mar 18;286(11):9840-8.
doi: 10.1074/jbc.M110.197079. Epub 2011 Jan 18.

Cardiac myocyte-specific ablation of follistatin-like 3 attenuates stress-induced myocardial hypertrophy

Masayuki Shimano  1 , Noriyuki Ouchi, Kazuto Nakamura, Yuichi Oshima, Akiko Higuchi, David R Pimentel, Kalyani D Panse, Enrique Lara-Pezzi, Se-Jin Lee, Flora Sam, Kenneth Walsh
Affiliations

Cardiac myocyte-specific ablation of follistatin-like 3 attenuates stress-induced myocardial hypertrophy

Masayuki Shimano et al. J Biol Chem. .

Abstract

Transforming growth factor-β family cytokines have diverse actions in the maintenance of cardiac homeostasis. Follistatin-like 3 (Fstl3) is an extracellular regulator of certain TGF-β family members, including activin A. The aim of this study was to examine the role of Fstl3 in cardiac hypertrophy. Cardiac myocyte-specific Fstl3 knock-out (KO) mice and control mice were subjected to pressure overload induced by transverse aortic constriction (TAC). Cardiac hypertrophy was assessed by echocardiography and histological and biochemical methods. KO mice showed reduced cardiac hypertrophy, pulmonary congestion, concentric LV wall thickness, LV dilatation, and LV systolic dysfunction after TAC compared with control mice. KO mice displayed attenuated increases in cardiomyocyte cell surface area and interstitial fibrosis following pressure overload. Although activin A was similarly up-regulated in KO and control mice after TAC, a significant increase in Smad2 phosphorylation only occurred in KO mice. Knockdown of Fstl3 in cultured cardiomyocytes inhibited PE-induced cardiac hypertrophy. Conversely, adenovirus-mediated Fstl3 overexpression blocked the inhibitory action of activin A on hypertrophy and Smad2 activation. Transduction with Smad7, a negative regulator of Smad2 signaling, blocked the antihypertrophic actions of activin A stimulation or Fstl3 ablation. These findings identify Fstl3 as a stress-induced regulator of hypertrophy that controls myocyte size via regulation of Smad signaling.

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Figures

FIGURE 1.
FIGURE 1.
Loss of Fstl3 attenuates exacerbation of pressure overload induced cardiac hypertrophy and heart failure. A and B, ratio of heart weight to body weight (HW/BW) (A) and lung weight to body weight (LW/BW) (B) in WT and cardiac myocyte-specific Fstl3 mice at 3 weeks after sham operation or TAC. C–F, echocardiographic analysis of diastolic intraventricular septum (IVSd) (C), diastolic posterior wall (PWd) (D), LV end systolic diameter (LVEDD) (E), and the fractional shortening (%FS) (F) for WT and KO mice at 3 weeks after sham operation or TAC. G, representative histological LV sections from WT or KO mice stained with Masson's trichrome. Heart samples were collected at 3 weeks after sham operation or TAC. Magnification, ×400. H and I, quantitative analysis of the CSA of cardiomyocytes (H) and the intestinal fibrosis area (I) from each group. Results are presented as mean ± S.E. For sham-treated mice, n = 6 for WT and KO. For TAC-treated mice, n = 12 for WT and n = 11 for KO. *, p < 0.05 versus corresponding sham; #, p < 0.05 versus corresponding WT mice.
FIGURE 2.
FIGURE 2.
Fstl3 deficiency leads to increased Smad2 activation following pressure overload. A and B, Fstl3 transcript and protein levels and activin A mRNA levels in the hearts of WT and KO mice at 1 week after sham operation or TAC. C, serum activin A protein levels for WT and KO mice at 0, 1, or 3 weeks after TAC. D, myostatin mRNA levels in the hearts of WT and KO mice at 1 week after sham operation or TAC. E, changes in phosphorylation of Smad2 (P-Smad2) in the hearts of WT and KO mice at 1 week after sham operation or TAC. Representative blots of phosphorylated and total Smad2 are shown. Lower panels show quantitative analysis of Smad2 phosphorylation. Relative phosphorylated levels of Smad2 were normalized to control values in sham-treated WT mice (n = 6–12 hearts in each group). *, p < 0.05 versus corresponding sham; #, p < 0.05 versus corresponding WT mice.
FIGURE 3.
FIGURE 3.
Fstl3 ablation attenuates the hypertrophic actions of PE treatment in cultured cardiac myocytes. A and B, results of real-time PCR and Western blot analysis of Fstl3 (A) and activin A (B) gene expression with unrelated or Fstl3 siRNA in the presence or absence of PE (100 μmol/liter) for 24 h. C and D, no effect of Fstl3 deletion on activin A mRNA and protein expression by NRVMs. Activin A protein levels in media were assessed by ELISA. E, effect of Fstl3 knockdown on Smad2 phosphorylation. Relative phosphorylated levels of Smad2 were normalized to control values in NRVMs that were not targeted with siRNA. F, representative fluorescence microscope images of NRVMs transfected with unrelated siRNA or Fstl3 siRNA for 48 h following by stimulation with phenylephrine (PE, 100 μmol/liter) for 24 h. G and H, quantitative analysis of cell surface area (G) and protein synthesis (H) assessed by [3H]leucine incorporation after siRNA transfection with or without PE. *, p < 0.05 versus corresponding control without PE; #, p < 0.05 versus corresponding no-targeting siRNA.
FIGURE 4.
FIGURE 4.
Antihypertrophic effects of Fstl3 knockdown are reversed by Smad7 transduction in cultured cardiac myocytes. A, quantitative analysis of cell surface area after siRNA and/or Ad-Smad7 transfection with or without PE. B–D, results of real-time PCR analysis of BNP (B), ANF (C), and sk-actin (D) expression following transduction of unrelated or Fstl3 siRNA in the presence or absence of Ad-Smad7 transduction. Some cells were treated with PE. Results are presented as mean ± S.E. (n = 8 per group). E, effect on Smad2 phosphorylation. Representative blots of phosphorylated and total Smad2 are shown. Lower panels show quantitative analysis of Smad2 phosphorylation. Relative phosphorylated levels of Smad2 were normalized to control values. *, p < 0.05 versus corresponding WT (both no-targeting siRNA and Ad-β-galactosidase (Ad-βgal)) without PE. #, statistical difference (p < 0.05) between indicated bars.
FIGURE 5.
FIGURE 5.
Antihypertrophic effect of Fstl3 knockdown can be reversed by ALK inhibition in cultured cardiac myocytes. A, quantitative analysis of cell surface area after siRNA transfection with or without PE or SB432541(10 μmol/liter). B–D, results of quantitative RT-PCR analysis of BNP (B), ANF (C), and sk-actin (D) expression in NRVMs treated with unrelated or Fstl3 siRNA in the presence or absence of PE or SB432541. E, effect on Smad2 phosphorylation. Representative blots of phosphorylated and total Smad2 are shown. Lower panels show quantitative analysis of Smad2 phosphorylation. Relative phosphorylated levels of Smad2 were normalized to control values. *, p < 0.05 versus corresponding WT without PE. #, statistical difference (p < 0.05) between indicated bars.
FIGURE 6.
FIGURE 6.
Fstl3 impairs activin A-mediated suppression of PE-induced cardiac hypertrophy in cultured cardiac myocytes. A, quantitative analysis of cell surface area after treatment with recombinant activin A protein (rActA, indicated dose or 100 ng/ml) with or without PE (100 μmol/liter) treatment. NRVMs were transfected with Ad-Fstl3, Ad-Smad7, or Ad-β-galactosidase (Ad-βgal). B–D, results of real-time PCR analysis of ANF (B), BNP (C), and sk-actin (D). E, effect on Smad2 phosphorylation. Representative blots of phosphorylated and total Smad2 are shown. Lower panels show quantitative analysis of Smad2 phosphorylation. Relative phosphorylated levels of Smad2 were normalized to control values. Cells were transduced with Ad-Fstl3 or Ad-Smad7 in the presence of PE inhibited to examine the regulatory activity of rActA. #, p < 0.05 versus corresponding Ad-β-galactosidase with rActA-treated cells. F, Western blot analysis revealed Fstl3 or Smad7 protein expression after transfection with Ad-Fstl3 or Ad-Smad7. G, model of Fstl3/Activin A-mediated regulation of cardiac hypertrophy.
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