Cardiac Myocyte-specific Ablation of Follistatin-like 3 Attenuates Stress-induced Myocardial Hypertrophy^*

Primary Neonatal Rat Ventricular Myocytes Culture

Primary culture of neonatal rat ventricular myocytes (NRVMs) were prepared as described previously (27). NRVMs were incubated in DMEM supplemented with 7% fetal calf serum (FCS) for 18 h after preparation and subsequently transfected with the rat small interfering RNA (siRNA) reagent, purchased from Dharmacon Inc., that represents a mixture of four different siRNAs that target Fstl3 (SMARTpool®). Transfection was performed by Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. Forty-eight hours after transfection, NRVMs were incubated with phenylephrine (PE, 100 μmol/liter). In some experiments, NRVMs were infected with adenoviral vectors expressing Fstl3 (Ad-Fstl3), Smad7 (Ad-Smad7), Smad2 (Ad-Smad2), or β-galactosidase (Ad-β-galactosidase) at a multiplicity of infection of 50 for 8 h in DMEM. The media were then replaced with fresh DMEM without adenovirus. Forty-eight hours after infection, NRVMs were incubated with PE (100 μmol/liter). In other experiments, serum-deprived NRVMs were incubated with recombinant activin A (100 ng/ml; Sigma) for 24 h. NRVMs were also preincubated with the activin receptor-like kinase (ALK) inhibitor, SB431542 (10 μmol/liter; Calbiochem) for inhibition of Smad2 phosphorylation for 1 h prior to activin A treatment.

Transverse Aortic Constriction (TAC) Protocol

Studies using cardiac myocyte specific Fstl3 knock-out mice (KO) in a C57BL/6 background were approved by the Institutional Animal Care and Use Committee of Boston University. As described previously (25), cardiac myocyte-specific Fstl3-deficient mice were generated by crossing Fstl3^flox/flox with transgenic mice expressing Cre-recombinase under the control of the α-myosin heavy chain promoter (The Jackson Laboratory) (28). We compared KO mice (Cre-Fstl3^flox/flox) with control (Cre-Fstl3^+/+) mice. Mice (8–10 weeks old) were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally). The chest was opened, and the thoracic aorta was identified after blunt dissection through the intercostal muscles. A 7–0 silk suture was placed around the transverse aorta and tied around a 26-gauge blunt needle, which was subsequently removed as described previously (4). Sham-operated mice underwent a similar surgical procedure without constriction of the aorta. Animals were anesthetized and then killed. The hearts and lungs were removed and weighed. In some experiments, heart and blood samples were extracted 7 days after TAC.

Echocardiography

After 21 days, we subjected surviving mice to transthoracic echocardiography to determine cardiac function and structure. To measure LV systolic function and chamber dimensions, we performed echocardiography with an Acuson Sequoia C-256 machine using a 15-MHz probe. After we obtained a two-dimensional image, we quantified diastolic interventricular septum, diastolic posterior wall thickness, LV end diastolic diameter, and percent LV fractional shortening from M-mode images.

Histology

Mice were killed, and LV tissue was obtained 21 days after the TAC procedure. Heart samples were embedded in OCT compound (Miles) and frozen in liquid nitrogen. Tissue slices (7 μm in thickness) were prepared. To determine the myocyte cell surface area (CSA) and the levels of myocardial interstitial fibrosis, sections stained with Masson's trichrome were quantified, as described previously (4).

Western Blot Analysis

Tissue samples obtained on postoperative day 7 were homogenized in lysis buffer (Cell Signaling Technology) with protease inhibitor mixture (Sigma). Protein content was determined by the Bradford method. Equal amounts of protein (100 μg) were separated in denaturing 8–12% polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were immunoblotted with primary antibodies at a 1:1,000–5,000 dilution followed by the secondary antibody conjugated with horseradish peroxidase (HRP) at a 1:1,000–10,000 dilution. Proteins were visualized using ECL Western blotting detection kit (Amersham Biosciences). To detect Fstl3 in heart, lysates were concentrated ∼10-fold using a Microcon device (Millipore) prior to analysis.

RNA Isolation and Quantitative RT-PCR

Total RNA was prepared from mouse heart by using an RNA isolation kit for fibrous tissue (Qiagen). Total RNA from cultured cells was prepared with an RNA isolation kit (Qiagen). Complementary DNA (cDNA) was synthesized from 500 ng of total RNA by using SuperScript RT-PCR Systems (Invitrogen). Quantitative real-time RT-PCR was carried out on an iCycler (Bio-Rad), using SYBR Green (Applied Biosystems). The primer sequences used were as follows: mouse: GAPDH forward (F), 5′-TCACCACCATGGAGAAGGC-3′ and reverse (R), 5′-GCTAAGCAGTTGGTGGTGCA-3′; Fstl3 F, 5′-CAACCCCGGCCAAGAACT-3′ and R, 5′-CTTCCTCCTCTGCTGGTACTTTG-3′; activin A F, 5′-TGGTGCCAGTCTAGTGCTTC-3′ and R, 5′-CCGTCACTCCCATCTTTCTT-3′; rat: GAPDH forward (F), 5′-TCAAGAAGGTGGTGAAGCAG-3′ and reverse (R), 5′-AGGTGGAAGAATGGGAGTTG-3′; Fstl3 F, 5′-CGTTACCTACATCTCGTCGTGT-3′ and R, 5′-TCTCTTCCTCCTCTGCTGGTA-3′; activin A F, 5′-ATGTGCGGATTGCTTGTG-3′ and R, 5′-CTTCCCGTCTCCATCCA-3′. The expression levels of examined transcripts were compared with those of GAPDH and normalized to the mean value of controls.

Analyses of Fstl3, Activin A, and Signaling in Hearts after TAC Injury

Levels of Fstl3 and activin A, a high affinity binding partner of Fstl3 (15, 18), were assessed in the sham and TAC-treated hearts. Consistent with our previous study (25), Fstl3 and activin A transcript levels in the hearts of control mice were up-regulated 9- and 3-fold, respectively, 1 week after pressure overload (Fig. 2, A and B). Protein levels of Fstl3 also increased in control mice, but this induction was absent in the KO mice after TAC (Fig. 2A), suggesting that the induction of Fstl3 expression by hypertrophic stimuli takes place primarily in cardiac myocytes. KO mice also displayed reductions in basal Fstl3 transcript and Fstl3 protein levels in sham hearts. Activin A transcript levels in heart and circulating protein levels increased after TAC, but no differences were found between KO and control mice (Fig. 2, B and C). Similarly, there were no differences in myostatin transcript levels in heart between the two strains (Fig. 2D).

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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.

To determine the effect of Fstl3 ablation on the possible participation of Smad signaling in pressure overload cardiac hypertrophy, the expression and phosphorylation of different Smad proteins at 1 week after TAC were assessed by Western blot analysis. As shown in Fig. 2E, the phosphorylation of Smad2 at Ser⁴⁶⁵/Ser⁴⁶⁷ did not differ under sham-operated conditions between KO and control mice or after TAC in control mice. However, Smad2 phosphorylation was increased in response to TAC in KO mice. In contrast, no changes were detected in the myocardial expression and/or phosphorylation of Smad1/5, Smad4, or Smad6 protein either between mouse strains or in response to TAC (supplemental Fig. 2). Therefore, the effect of Fstl3 ablation appeared to specifically affect Smad2 signaling under conditions of pressure overload.

Ablation of Fstl3 Inhibits Cardiac Myocyte Growth in Vitro

To examine the role of Fstl3 in cardiac growth at a mechanistic level, an in vitro model of cardiac myocyte hypertrophy using PE and siRNA-mediated down-regulation of Fstl3 expression was employed. PE treatment increased the transcript levels and protein of Fstl3 and activin A in NRVMs (Fig. 3, A–D). Transfection of NRVMs with siRNA against Fstl3 resulted in a 69% decrease in Fstl3 mRNA levels under basal conditions and 79% under PE stimulation conditions (Fig. 3A). In contrast, Fstl3 knockdown did not alter basal or PE-induced activin A expression at the mRNA (Fig. 3C) or protein (Fig. 3D) levels, but Smad2 phosphorylation was increased in response to PE in the Fstl3 knockdown condition (Fig. 3E). Knockdown of Fstl3 did not affect myocyte CSA under basal conditions, but it inhibited the PE-mediated increase in CSA (Fig. 3, F and G). The ablation of Fstl3 in cardiomyocytes markedly attenuated PE-stimulated protein synthesis as assessed by [³H]leucine uptake (Fig. 3H). Fstl3 knockdown also inhibited PE-induced brain natriuretic peptide (BNP), atrial natriuretic factor (ANF), and α-skeletal actin (sk-actin) expression (Fig. 4).

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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.

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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.

To explore the role of Smad signaling in Fstl3 modulation of stress-induced cardiac hypertrophy, a series of experiments were performed with an adenoviral vector expressing Smad7, an inhibitor of Smad2 activity (29). As shown in Fig. 4A, transduction with Smad7 reversed the inhibitory action of Fstl3 knockdown on the PE-induced increase in myocyte CSA. Consistent with these data, treatment with 10 μm SB431542, a specific inhibitor of TGF-β superfamily type I ALK receptors (ALK4, 5, and 7) (30), blocked the inhibition of PE-induced myocytes hypertrophy by Fstl3 knockdown (Fig. 5A). The attenuation of PE-stimulated cardiac marker expression (ANF, BNP, and sk-actin) by Fstl3 ablation was also reversed by transduction with Ad-Smad7 (Fig. 4, B–D). Similarly, treatment with SB431542 also reversed the suppressive actions of Fstl3 knockdown on PE-induced BNP, ANF, and sk-actin expression in cultured myocytes (Fig. 5, B–D). As shown in Figs. 4E and ​and55E, both SB431542 and Smad7 abolished PE-stimulated Smad2 phosphorylation. Collectively, these data suggest that Fstl3 modulates stress-induced hypertrophy via regulation of ALK-mediated Smad signaling.

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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.