Transforming growth factor betas are upregulated in the rat masseter muscle hypertrophied by clenbuterol, a beta2 adrenergic agonist

Abstract

1. The regulatory mechanism for the hypertrophy of skeletal muscles induced by clenbuterol is unclear. The purpose of the present study was to determine the extent to which transforming growth factor betas (TGFbetas), fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), and platelet-derived growth factors (PDGFs) are involved in the hypertrophy of rat masseter muscle induced by clenbuterol. 2. We measured the mRNA expression levels for TGFbetas, FGFs, HGF, and PDGFs in rat masseter muscle hypertrophied by oral administration of clenbuterol for 3 weeks and determined correlations between the weight of masseter muscle and mRNA expression levels by regression analysis. We determined immunolocalizations of TGFbetas and their receptors (TGFbetaRs). 3. The mRNA expression levels for TGFbeta1, 2, and 3, and for PDGF-B demonstrated clenbuterol-induced elevations and positive correlations with the weight of masseter muscle. In particular, TGFbeta1, 2, and 3 showed strong positive correlations (correlation coefficients >0.6). The mRNA expression levels for PDGF-A, FGF-1 and 2, and HGF showed no significant differences between the control and clenbuterol groups, and no significant correlations. TGFbeta1, 2, and 3 were principally localized in the connective tissues interspaced among myofibers, and TGFbetaRI and II were localized in the periphery and sarcoplasm of the myofibers. 4. These results suggest that paracrine actions of TGFbeta1, 2, and 3 via TGFbetaRI and II could be involved in the hypertrophy of rat masseter muscle induced by clenbuterol. This is the first study to document the involvement of TGFbetas in the hypertrophy of skeletal muscles induced by clenbuterol.

Figure 1

(a) Changes in body weight of rats in the control group (solid line) and clenbuterol group (dotted line) during the clenbuterol administration. Each point and its vertical bar represent the mean±1 s.d. of nine rats. (b, c) Bar graphs showing the wet weight of left masseter muscle (b) and the fiber diameter of right masseter muscle (c) in the control group (Cont) and clenbuterol group (Clen) at 3 weeks from the initiation of clenbuterol administration. Each column and its vertical bar represent the mean+1 s.d. of nine rats. Significant difference between the control and clenbuterol groups, ^*P<0.05, ^***P<0.001.

Figure 2

(a) Gel electrophoretic pattern for transforming growth factor β (TGFβ) 1 competitive RT–PCR products of the left masseter muscle of the control (Cont) and clenbuterol (Clen) groups. The target gene is shown in the upper bands, while the competitor is shown in the lower band. (b–d) Bar graphs showing the mRNA expression levels for TGFβ1 (b), 2 (c), and 3 (d) in the left masseter muscle of the control and clenbuterol groups. Each column and vertical bar represent the mean+1 s.d. of nine rats. The vertical axis is expressed as a percentage of the mean control value set at 100. Significant differences between the control and clenbuterol groups, ^*P<0.05; ^***P<0.001.

Figure 3

(a–c) Scatter diagrams and regression lines between the weight of left masseter muscle and the mRNA expression levels for TGFβ1 (a), 2 (b), and 3 (c). The vertical axis is expressed as a percentage of the mean control value set at 100. Values for both the control and clenbuterol groups were included.

Figure 4

(a–e) Bar graphs showing the mRNA expression levels for platelet-derived growth factor (PDGF)-A (a) and B (b), fibroblast growth factor (FGF)-1 (c) and 2 (d), and hepatocyte growth factor (HGF) (e) in the left masseter muscle of the control (Cont) and clenbuterol (Clen) groups. Each column and vertical bar represent the mean+1 s.d. of nine rats. The vertical axis is expressed as a percentage of the mean control value set at 100. Significant difference between the control and clenbuterol groups, ^**P<0.01.

Figure 5

(a–e) Scatter diagrams and regression lines between the weight of left masseter muscle and the mRNA expression levels for PDGF-A (a) and B (b), FGF-1 (c) and 2 (d), and HGF (e). The vertical axis is expressed as a percentage of the mean control value set at 100. Values for both the control and clenbuterol groups were included.

Figure 6

(a–f) Immunolocalization for TGFβ1 (a, b), 2 (c, d), and 3 (e, f) in the middle portions of the right masseter muscle in the control (a, c, e) and clenbuterol (b, d, f) rats. Arrows in (a and b), and those in (e and f) indicate the regions intensively immunostained for TGFβ1 and TGFβ3, respectively. (b–f) are at the same magnification as that in (a).

Figure 7

(a–d) Immunolocalization for transforming growth factor β receptor (TGFβRI) (a, b) and II (c, d) in the middle portions of the right masseter muscle in the control (a, c) and clenbuterol (b, d) rats. (b–d) are at the same magnification as that in (a).