IGF-1 expression in infarcted myocardium and MGF E peptide actions in rat cardiomyocytes in vitro

Abstract

Insulinlike growth factor-1 (IGF-1) expression is implicated in myocardial pathophysiology, and two IGF-1 mRNA splice variants have been detected in rodents, IGF-1Ea and mechano-growth factor (MGF). We investigated the expression pattern of IGF-1 gene transcripts in rat myocardium from 1 h up to 8 wks after myocardial infarction induced by left anterior descending coronary artery ligation. In addition, we characterized IGF-1 and MGF E peptide action and their respective signaling in H9C2 myocardial-like cells in vitro. IGF-1Ea and MGF expression were significantly increased, both at transcriptional and translational levels, during the late postinfarction period (4 and 8 wks) in infarcted rat myocardium. Measurements of serum IGF-1 levels in infarcted rats were initially decreased (24 h up to 1 wk) but remained unaltered throughout the late experimental phase (4 to 8 wks) compared with sham-operated rats. Furthermore, specific anti-IGF-1R neutralizing antibody failed to block the synthetic MGF E peptide action, whereas it completely blocked IGF-1 action on the proliferation of H9C2 cells. Moreover, this synthetic MGF E peptide did not activate Akt phosphorylation, whereas it activated ERK1/2 in H9C2 rat myocardial cells. These data support the role of IGF-1 expression in the myocardial repair process and suggest that synthetic MGF E peptide actions may be mediated via an IGF-1R independent pathway in rat myocardial cells, as suggested by our in vitro experiments.

Figure 1

Histopathological analysis of the size of the infarcted region in rat myocardium using the Tetrazolium (TTC) method. Viable myocardium was stained brick red, whereas infarcted myocardium failed to be stained with TTC. In our experiments, 20%–40% of the rat myocardium was apparently damaged. Presented here is an example of rat myocardium excised and cut from base (upper panel) to apex (lower panel) transversely in 2-mm thick sections and stained with TTC.

Figure 2

Expression of the IGF-1 isoforms in rat myocardium. Panel I: Specific PCR products for MGF and IGF-1Ea mRNA detected in rat myocardium. The PCR products were confirmed by sequencing. Panel II: Detection of MGF and IGF-1Ea at the protein level using Western blot analysis. Panel III: Photomicrographs (magnification, × 40) of the left ventrical sections of rat myocardium stained with anti-MGF antibody. Specificity of the immunohistochemical MGF detections was confirmed by the absence of immunoreactivity in the negative control sections (A). Note that myocardial tissues of sham-operated rats appear less intensively stained (B) compared with infarcted tissues (C).

Figure 3

Panel I: mRNA expression of IGF-1Ea and MGF splice variants at different time points after experimental occlusion of the left descending coronary artery. Bars represent mean values (mean ± SEM of six measurements) of respective mRNA expression, which was normalized to each corresponding ribosomal 18S and expressed as the fold change of the mRNA levels in sham-operated rats (controls). PCR amplification for both 18S and the different IGF-1 transcripts was in linear phase at 37 cycles at all time points. *Statistically significant values: P < 0.05. Panel II: Expression of IGF-1Ea and MGF at the protein level using Western blot analysis. Immunoblotting for GAPDH served as a control for protein loading. Expression values (means ± SEM of six measurements) were normalized to those of corresponding GAPDH in the same immunoblot and expressed as the fold change of the sham-operated rats (controls). *Statistically significant values: P < 0,05.

Figure 4

Serum IGF-1 levels measured in sham-operated and infarcted rats using enzyme-linked immunosorbent assay as described in the “Material and Methods” section. Concentrations of IGF-1 were expressed as mean ± SEM of six measurements.*P < 0.05. Note that IGF-1 serum levels were decreased early (1 h to 1 wk) during the experimental procedure; however, these levels were not different during the late phase (4 wks and 8 wks) of the experimental procedure compared with measurements in sham-operated rats.

Figure 5

Analysis of H9C2 cell proliferation using the trypan blue assay. Panel I: The effects of mature IGF-1 and synthetic MGF E peptide on H2C2 cell proliferation (% cell number changes) in a dose-dependent manner as presented in the “Materials and Methods” section. Note that the IGF-1R neutralizing antibody (anti-IGF-1R ab) blocked IGF-1 effects without affecting the action of synthetic MGF E peptide on H9C2 cell proliferation. Data are presented as mean ± SEM. *Significantly different (P < 0.001) from control conditions. Panel II: Representative Western blot analysis of ERK1/2 and Akt phosphorylation in H9C2 cells after stimulation with IGF-1 and synthetic MGF E peptide in a time-dependent manner. Note that the MGF E peptide activated ERK1/2 phosphorylation; however, it did not activate Akt phosphorylation. IGF-1 did activate both ERK1/2 and Akt in H9C2 myocardial-like cells as expected. These data suggest that synthetic MGF E peptide could signal via an IGF-1R–independent mechanism in H9C2 cells.