1. In Cardiomyocyte Hypoxia, Insulin-Like Growth Factor-I-Induced Antiapoptotic Signaling Requires Phosphatidylinositol-3-OH-Kinase-Dependent and Mitogen-Activated Protein Kinase-Dependent Activation of the Transcription Factor cAMP Response Element-Binding Protein
by Felix B. Mehrhof, Frank- Ulrich Müller, Martin W. Bergmann, Peifeng Li, Yibin Wang, Wilhelm Schmitz, Rainer Dietz, and Rüdiger von Harsdorf
Circulation
Volume 104(17):
October 23, 2001
Copyright © American Heart Association, Inc. All rights reserved.

2. Figure 1. IGF-I inhibits hypoxia-induced apoptosis of neonatal cardiomyocytes in a PI3K- and MEK1-dependent manner.
Figure 1. IGF-I inhibits hypoxia-induced apoptosis of neonatal cardiomyocytes in a PI3K- and MEK1-dependent manner. A, Time course and dose dependence of cell survival effects of IGF-I on cardiomyocytes. Cells were cultured in normoxia or hypoxia for the indicated times and supplemented with different doses of IGF-I. Dead cells were counted by the trypan blue exclusion assay. B, Analysis of DNA fragmentation. Cells were cultured in normoxia for 24 hours (1), hypoxia for 24 hours (2), or hypoxia+IGF-I (100 ng/mL) for 24 hours (3) followed by extraction and electrophoresis of DNA. M indicates marker. C, Quantification of apoptotic cardiomyocytes by TUNEL staining and α-sarcomeric actin counterstaining after normoxia for 24 hours, hypoxia for 24 hours, or hypoxia+IGF-I (100 ng/mL) for 24 hours. At least 20 cardiomyocyte nuclei were counted in 10 random fields per experiment (n=3). *P<0.01 vs control; #P<0.05 vs hypoxia. D, Quantitative analysis of apoptosis by cell death ELISA. Cells were cultured in normoxia or hypoxia for 36 hours with or without supplementation of IGF-I (100 ng/mL). When indicated, pharmacological inhibitors (WM indicates wortmannin 20 nmol/L; PD, PD μmol/L; and SB, SB μmol/L) were added to the culture medium 15 minutes before IGF-I. Pooled data from 4 independent experiments are shown. *P<0.01 vs hypoxia alone, #P<0.05 vs hypoxia in the presence of IGF-I.
Felix B. Mehrhof et al. Circulation. 2001;104:
Copyright © American Heart Association, Inc. All rights reserved.

3. Figure 2. AKT and ERK1 and ERK2 are phosphorylated in response to IGF-I.
Figure 2. AKT and ERK1 and ERK2 are phosphorylated in response to IGF-I. Western blots with (bottom lanes) or without (top lanes) phospho-specific antibodies and quantitative analysis are shown (mean±SEM). A and B, Cells were cultured in normoxia or hypoxia for 24 hours with or without IGF-I-supplementation. Inhibitors (wortmannin 20 nmol/L [WM], PD μmol/L [PD], and SB μmol/L [SB]) were added 15 minutes before IGF-I (100 ng/mL) (n=3; *P<0.05 vs hypoxia alone; #P<0.001 vs hypoxia+IGF-I). C, Influence of different doses of wortmannin on IGF-I-mediated ERK1 and ERK2 phosphorylation. Cells were cultured in normoxia and stimulated with IGF-I (100 ng/mL) for 1 hour (n=3; *P<0.05 vs control). D, Influence of different doses of wortmannin on IGF-I-mediated AKT phosphorylation. Cellular extracts were from the same cells as in panel C (n=3; *P<0.05 vs control).
Felix B. Mehrhof et al. Circulation. 2001;104:
Copyright © American Heart Association, Inc. All rights reserved.

4. Figure 3. Both IGF-I and hypoxia induce phosphorylation of BAD at serine residues S112 and S136.
Figure 3. Both IGF-I and hypoxia induce phosphorylation of BAD at serine residues S112 and S136. A, Cells were cultured in normoxia and treated with IGF-I (100 ng/mL) and inhibitors (wortmannin 20 nmol/L [WM] and PD μmol/L [PD]) for the indicated times. Immunoprecipitation with BAD antibody was followed by Western blot analysis. B, Cells were cultured in hypoxia for the indicated times, and phosphorylation of serine residues S112 and S136 was detected as in panel A. C, Analysis of the subcellular localization of BAD in cells exposed to different episodes of hypoxia. BAD was detected by immunoprecipitation and Western blot analysis of different fractions of cellular extracts. Densitometric analysis of blots is shown above (mean±SEM, n=3, *P<0.05 vs control).
Felix B. Mehrhof et al. Circulation. 2001;104:
Copyright © American Heart Association, Inc. All rights reserved.

5. Figure 4. IGF-I mediates protective effects by PI3K- and MEK1-dependent phosphorylation of CREB. A, CREB phosphorylation is analyzed using a phospho-specific antibody.
Figure 4. IGF-I mediates protective effects by PI3K- and MEK1-dependent phosphorylation of CREB. A, CREB phosphorylation is analyzed using a phospho-specific antibody. Cells were cultured in a hypoxic atmosphere for the indicated times and treated with IGF-I (100 ng/mL) and inhibitors (wortmannin 20 nmol/L [WM] and PD μmol/L [PD]) as indicated. Densitometric analysis of blots is shown above (mean±SEM, n=3, *P<0.05 vs control; #P<0.05 vs IGF-I-treatment). B, Quantitative analysis of apoptosis by cell death ELISA. Cells were infected with Ad-dnCREB or Ad-lacZ and treated as indicated. Pooled data from 3 independent experiments (*P<0.05 vs hypoxia+IGF-I).
Felix B. Mehrhof et al. Circulation. 2001;104:
Copyright © American Heart Association, Inc. All rights reserved.

6. Figure 5. IGF-I induces CREB-dependent expression of bcl-2.
Figure 5. IGF-I induces CREB-dependent expression of bcl-2. Cardiomyocytes were treated with IGF-I, inhibitors, and adenoviruses as indicated, and bcl-2 was detected by Western blot analysis. Tropomyosin Western blot was performed with the same extracts to demonstrate equal amounts of protein in all lanes. Densitometric analysis of blots is shown above (mean±SEM, n=3, *P<0.05 vs control; #P<0.05 vs hypoxia+24-hour IGF-I).
Felix B. Mehrhof et al. Circulation. 2001;104:
Copyright © American Heart Association, Inc. All rights reserved.

7. Figure 6. Simplified scheme for IGF-I-induced and hypoxia-induced signaling regulating survival of cardiomyocytes.
Figure 6. Simplified scheme for IGF-I-induced and hypoxia-induced signaling regulating survival of cardiomyocytes.
Felix B. Mehrhof et al. Circulation. 2001;104:
Copyright © American Heart Association, Inc. All rights reserved.