Inhibition of transforming growth factor-beta signaling induces left ventricular dilation and dysfunction in the pressure-overloaded heart - PubMed (original) (raw)

Inhibition of transforming growth factor-beta signaling induces left ventricular dilation and dysfunction in the pressure-overloaded heart

Jason A Lucas et al. Am J Physiol Heart Circ Physiol. 2010 Feb.

Abstract

This study utilized a transgenic mouse model that expresses an inducible dominant-negative mutation of the transforming growth factor (TGF)-beta type II receptor (DnTGFbetaRII) to define the structural and functional responses of the left ventricle (LV) to pressure-overload stress in the absence of an intact TGF-beta signaling cascade. DnTGFbetaRII and nontransgenic (NTG) control mice (male, 8-10 wk) were randomized to receive Zn(2+) (25 mM ZnSO(4) in drinking H(2)O to induce DnTGFbetaRII gene expression) or control tap H(2)O and then further randomized to undergo transverse aortic constriction (TAC) or sham surgery. At 7 days post-TAC, interstitial nonmyocyte proliferation (Ki67 staining) was greatly reduced in LV of DnTGFbetaRII+Zn(2+) mice compared with the other TAC groups. At 28 and 120 days post-TAC, collagen deposition (picrosirius-red staining) in LV was attenuated in DnTGFbetaRII+Zn(2+) mice compared with the other TAC groups. LV end systolic diameter and end systolic and end diastolic volumes were markedly increased, while ejection fraction and fractional shortening were significantly decreased in TAC-DnTGFbetaRII+Zn(2+) mice compared with the other groups at 120 days post-TAC. These data indicate that interruption of TGF-beta signaling attenuates pressure-overload-induced interstitial nonmyocyte proliferation and collagen deposition and promotes LV dilation and dysfunction in the pressure-overloaded heart, thus creating a novel model of dilated cardiomyopathy.

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Figures

Fig. 1.

A: expression of transforming growth factor-β type II receptor (DnTGFβRII) mRNA in tissues of DnTGFβRII mice drinking 25 mM ZnSO4 water (Zn2+; lanes 3–11) or double-distilled water (H2O; lanes 12–14) for 1 wk. RNA was isolated from snap-frozen tissue using TRIzol reagent (Invitrogen). For RT-PCR analysis, RNA was treated with RNase-free DNase for 30 min at 37°C to remove contaminating genomic DNA. cDNA was synthesized from 1 μg of total tissue RNA using random primers. PCR amplification of DnTGFβRII cDNA was performed using primers specific for the DnTGFβRII allele (5′-ATC-GTC-ATC-GTC-TTT-GTA-GTC-3′ and 5′-TCC-CAC-CGC-ACG-TTC-AGA-AG-3′). Genomic DNA was used as the PCR control (lanes 1 and 2). B: expression of FLAG epitope protein in left ventricle (LV) of DnTGFβRII mice drinking Zn2+ (lanes 1 and 2) or distilled water (lanes 3 and 4) for 1 wk. The FLAG protein gene is fused with the DnTGFβRII gene and coexpressed with DnTGFβRII protein with Zn2+ stimulation as described by Serra et al. (31). C: effects of 1 wk transverse aortic constriction (TAC) on phospho-Smad3 levels in LV of DnTGFβRII mice drinking Zn2+ or distilled water. Western immunoblotting was performed with selective anti-phospho-Smad antibody, and α-tubulin was used as an internal control for quantitation. Results are means ± SE; n = number of mice per group.

Fig. 2.

Representative light micrographs of LV from DnTGFβRII and nontransgenic (NTG) mice drinking 25 mM ZnSO4 water (Zn2+) or double-distilled water (H2O) 7 days after TAC or sham (control) operation. Cross sections of middle circular layer of posterior wall were immunostained with selective anti-nuclear Ki67 antibody (brown color). Arrows indicate representative positive signals localizing the interstitial nonmyocytes. Magnification = ×400.

Fig. 3.

Effects of 7 days of TAC or sham operation (control) on density of Ki-67 positive interstitial nonmyocytes in posterior wall (A) and septum (B) of DnTGFβRII and NTG mice drinking 25 mM ZnSO4 water (Zn2+) or distilled water (H2O). Six ×400 cross-sectional areas of posterior wall and six septal areas per mouse were measured and averaged. Results are means ± SE; n = number of mice per group. *P < 0.05 compared with respective sham control groups; #P < 0.05 compared with respective TAC-H2O groups by ANOVA.

Fig. 4.

Representative picrosirius red-stained cross sections at a level below the mitral valve of posterior wall of DnTGFβRII and NTG mice drinking 25 mM ZnSO4 water (Zn2+) or distilled water (H2O) 28 and 120 days after TAC. Controls were sham-operated mice without TAC and killed at 14–16 wk of age. Magnification = ×400.

Fig. 5.

Effects of 28 and 120 days of TAC on interstitial collagen volume (picrosirius red-stained areas) in LV of DnTGFβRII and NTG mice drinking 25 mM ZnSO4 water (Zn2+) or distilled water (H2O). Six ×400 cross section areas of posterior wall and six septal areas per mouse were measured and averaged. Controls are mice without TAC and killed at 14–16 wk of age. Results are means ± SE; n = number of mice per group. *P < 0.05 compared with respective sham control groups; #P < 0.05 compared with respective TAC-H2O groups by ANOVA.

Fig. 6.

Representative micrographs of 2-D guided M-Mode echocardiography of Sham-NTG+H2O, TAC-NTG+H2O, and TAC-DnTGFBRII+Zn2+ mice at 120 days after TAC or sham operation. Double-head arrows indicate LV end-diastolic dimension (LVEDD).

Fig. 7.

Effects of 120 days of TAC on body weight (BW: A), LV/BW ratio (B), LVEDD (C), left ventricular end systolic dimension (LVESD; D), end diastolic volume (EDV; E), end systolic volume (ESV; F), ejection fraction (EF; G), and fractional shortening (FS; H) in DnTGFBRII and NTG mice drinking 25 mM ZnSO4 water (Zn2+) or distilled water (H2O). Controls are mice drinking H2O and killed at the same age as TAC mice. EDV, ESV, EF, and FS were assessed by echocardiography and adjusted by ANOVA with BW as a covariate. Results are means ± SE; n = number of mice per group. *P < 0.05 compared with sham control groups; #P < 0.05 compared with respective H2O-TAC groups; Δ_P_ < 0.05 compared with respective NTG groups by ANOVA.

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Inhibition of transforming growth factor-beta signaling induces left ventricular dilation and dysfunction in the pressure-overloaded heart - PubMed (original) (raw)