Cardiac myocyte follistatin-like 1 functions to attenuate hypertrophy following pressure overload

Abstract

Factors secreted by the heart, referred to as "cardiokines," have diverse actions in the maintenance of cardiac homeostasis and remodeling. Follistatin-like 1 (Fstl1) is a secreted glycoprotein expressed in the adult heart and is induced in response to injurious conditions that promote myocardial hypertrophy and heart failure. The aim of this study was to investigate the role of cardiac Fstl1 in the remodeling response to pressure overload. Cardiac myocyte-specific Fstl1-KO mice were constructed and subjected to pressure overload induced by transverse aortic constriction (TAC). Although Fstl1-KO mice displayed no detectable baseline phenotype, TAC led to enhanced cardiac hypertrophic growth and a pronounced loss in ventricular performance by 4 wk compared with control mice. Conversely, mice that acutely or chronically overexpressed Fstl1 were resistant to pressure overload-induced hypertrophy and cardiac failure. Fstl1-deficient mice displayed a reduction in TAC-induced AMP-activated protein kinase (AMPK) activation in heart, whereas Fstl1 overexpression led to increased myocardial AMPK activation under these conditions. In cultured neonatal cardiomyocytes, administration of Fstl1 promoted AMPK activation and antagonized phenylephrine-induced hypertrophy. Inhibition of AMPK attenuated the antihypertrophic effect of Fstl1 treatment. These results document that cardiac Fstl1 functions as an autocrine/paracrine regulatory factor that antagonizes myocyte hypertrophic growth and the loss of ventricular performance in response to pressure overload, possibly through a mechanism involving the activation of the AMPK signaling axis.

Fig. 1.

Expression of Fstl1 in heart. (A and B) Changes in Fstl1 transcript (A) and protein (B) in the hearts of WT and cardiac myocyte-specific Fstl1-KO mice 1 wk after TAC or sham surgery. Representative blots of Fstl1 and GAPDH are shown in B. (C) Fstl1 protein levels in isolated cardiac myocytes. Western blot analysis was performed to evaluate Fstl1 protein expression in cardiac myocytes isolated from WT or KO mice. (D) Fstl1 protein levels in serum from WT and KO mice 1 wk after TAC or sham surgery. A representative blot for Fstl1 is shown. (E) Immunochemical staining of Fstl1 in cardiac sections from WT or KO mice 4 wk after sham or TAC operation. Myocardial tissues from WT or KO mice were fixed and incubated with Fstl1 antibody, followed by incubation with biotin-conjugated secondary antibody and HRP-avidin. (F) Immunofluorescence of Fstl1 (green; Alexa Fluor 488) and α-actinin (red; Alexa Fluor 594) in cardiac sections from WT or KO mice 4 wk after sham or TAC operation. Blue is DAPI. *P < 0.05 versus corresponding sham-treated mice; ^#P < 0.05 versus corresponding WT mice.

Fig. 2.

Loss of Fstl1 expression in myocytes promotes pressure overload-induced cardiac hypertrophy and heart failure. (A and B) HW/BW (A) and LW/BW (B) ratios in WT and cardiac myocyte-specific Fstl1-KO mice 4 wk after TAC or sham surgery. (C–F) Echocardiographic parameters in different experimental groups of mice. Shown are measurements of IVSd (C), PWd (D), LVEDD (E), and %FS (F) for WT and KO mice 4 wk after TAC or sham surgery. For sham-treated mice, n = 8 WT mice and 8 KO mice. For TAC-treated mice, n = 16 WT mice and 14 KO mice. *P < 0.05 versus corresponding sham; ^#P < 0.05 versus corresponding WT mice.

Fig. 3.

Fstl1 deficiency in cardiac myocytes promotes TAC-induced myocyte hypertrophy and attenuates AMPK activation. (A) Representative histological sections from the left ventricle of WT and KO mice stained with Masson's trichrome. Heart samples were collected and processed 4 wk after TAC or sham surgery. (Magnification, 400x.) (B and C) Quantitative analysis of the CSA of cardiomyocytes (B) and intestinal fibrosis area (C) from each experimental group. Results are presented as mean ± SEM (n = 6–12 mice per group). (D and E) The results of qRT-PCR analysis of BNP (D) and ANF (E) expression. (F) Phosphorylation of AMPK (P-AMPK) at Thr172 in the hearts of WT and KO mice 1 wk after TAC or sham surgery. (Upper) Representative blots of phosphorylated and total AMPK. (Lower) Quantitative analysis of AMPK phosphorylation. Relative levels of AMPK phosphorylation were normalized to control values in sham-treated WT mice. n = 8 samples per group. *P < 0.05 versus corresponding sham-treated mice; ^#P < 0.05 versus WT mice.

Fig. 4.

Fstl1 overexpression protects against the development of cardiac hypertrophy and heart failure following pressure overload. (A) Western blot analysis was performed to evaluate Fstl1 protein expression in whole heart or serum in WT or striated muscle-specific Fstl1-TG mice. (B and C) HW/BW (B) and LW/BW (C) ratios in WT and Fstl1-TG mice 4 wk after TAC or sham surgery. (D–G) Echocardiographic measurements of IVSd (D), PWd (E), LVEDD (F), and %FS (G) for WT and TG mice 4 wk after TAC or sham surgery. For sham-treated mice, n = 6 WT and 6 TG mice. For TAC-treated mice, n = 12 WT mice and 13 TG mice. *P < 0.05 versus corresponding sham-treated mice; ^#P < 0.05 versus corresponding WT mice.

Fig. 5.

Gain of Fstl1 function attenuates myocyte hypertrophy and fibrosis and promotes AMPK activation in response to TAC. (A) Representative histological left ventricle sections from WT and TG mice treated with Masson trichrome stain. Heart samples were collected 4 wk after TAC or sham surgery. (Magnification, 400×.) (B and C) Quantitative analysis of the cardiomyocyte CSA (B) and intestinal fibrosis (C) from each experimental group. Results are presented as mean ± SEM (n = 6–12 mice per group). (D and E) The results of qRT-PCR analysis of BNP (D) and ANF (E) expression. (F) P-AMPK in the hearts of WT and TG mice 1 wk after TAC or sham surgery. (Upper) Representative blots of phosphorylated and total AMPK. (Lower) Quantitative analysis of AMPK phosphorylation. Relative phosphorylated levels of AMPK were normalized to control values in sham-treated WT mice (n = 8 hearts per group). *P < 0.05 versus corresponding sham-treated mice; ^#P < 0.05 versus WT mice.

Fig. 6.

Fstl1 attenuates the hypertrophic actions to PE treatment in cultured cardiac myocytes. (A) Quantitative analysis of PE-induced hypertrophy following transduction with adenoviral vector Ad-Fstl1 or Ad-βgal (control). Cultures of NRVMs were transduced at an MOI of 50 for 8 h. After 36 h of further incubation in fresh medium, cells were treated with PE for 24 h. (B and C) qRT-PCR analysis of BNP (B) and ANF (C) expression in cells transduced with Ad-Fstl1 or Ad-βgal in the presence or absence of PE (100 μmol/L) for 24 h. (D) Quantitative analysis of myocyte CSA after treatment with rFstl1 and PE. *P < 0.05 versus corresponding control; ^#P < 0.05 versus control treatment.

Fig. 7.

Inhibition of PE-stimulated myocyte hypertrophy by Fstl1 is mediated by AMPK signaling. Time-dependent (A) and dose-dependent (B) changes in the phosphorylation of AMPK after rFstl1 treatment in NRVMs. (A and B, Upper) Representative blots of phosphorylated and total ACC and AMPK are shown. (Lower) Quantitative analysis of AMPK phosphorylation. (C) Quantitative analysis of CSA after infection with Ad-dnAMPK or Ad-βgal (control). After vector transduction for 8 h, cells were washed and incubated for 36 h before treatment with PE (100 μmol/L) in the presence or absence of rFstl1 (250 ng/mL). *P < 0.05 versus corresponding control; ^#P < 0.05 versus Fstl1 treatment.

Fig. P1.

Ablation of Fstl1 in myocytes promotes cardiac hypertrophy and heart failure under cardiac pressure overload. (A) Induction of Fstl1 (green) expression in cardiac myocytes (shown in red by a myocyte-specific marker) in response to transverse aortic constriction (TAC). Colocalization (yellow) of Fstl1 and the myocyte-specific marker can be seen in WT but not in Fstl1-KO mice. Fstl1 expression in nonmyocytes also can be observed and is particularly evident in sections from Fstl1-KO mice. (B and C) Ratio of heart weight to body weight (HW/BW) (B) and lung weight to body weight (LW/BW) (C) in WT and Fstl1-KO mice at 4 wk after TAC or sham surgery. (D) Percent fractional shortening (%FS) determined from echocardiographic measurements for WT and Fstl1-KO mice at 4 wk after TAC or sham surgery. *P < 0.05 versus corresponding sham surgery; ^#P < 0.05 versus corresponding WT mice.