Autophagy fine-tuning by angiotensin-(1-9) in cultured rat cardiomyocytes

Figures

• 

  Figure 1. Angiotensin-(1-9) impairs autophagy in neonatal rat ventricular cultured cardiomyocytes. Cardiomyocytes in low serum were treated with 10 μM Ang-(1-9) for 3 and 6 h. Protein levels of LC3-II (A) and p62 (B) were monitored by Western blot and quantified. The graphs represent the average ± SEM of n = 3. No statistical significance was achieved for LC3-II and p62 levels. (C) Cardiomyocytes were treated for 24 h with 10 μM Ang-(1-9) in the presence or absence of 30 μM CQ for the last 4 h. Protein levels of LC3-II and total LC3 were monitored by Western blot and quantified. The graphs represent the average ± SEM of n = 5. (D) Total RNA was purified from cardiomyocytes treated or untreated with 10 μM Ang-(1-9) for 24 h. cDNA was synthesized and Lc3b, Bcn1, Atg5, Atg12 and Gabarapbl1 (Gab) genes were amplified though PCR. Endogenous Ywas and Hmbs genes were used as controls (n = 4). **p < 0.01, ***p < 0.001 CQ vs. basal; #p < 0.05 Ang-(1-9) vs. NT. NT, non-treated.

• 

  Figure 2. Angiotensin-(1-9) blocks angiotensin II-induced autophagy in neonatal rat ventricular cultured cardiomyocytes. Cardiomyocytes were cultured by 24 h in the presence of different concentrations of Ang-II (10–500 nM) and levels of LC3-II (A) and p62 (B) were determined through Western-blot and quantified. The graphs represent the average ± SEM. (C) Cells were cultured by 24 h with 10 nM Ang II in the presence of different concentrations of Ang-(1-9) (10–100 μM), and LC3-II levels were determined by Western-blot (n = 5). (D) LC3-II and p62 protein levels at cardiomyocytes cultured by 24 h with 10 nM Ang-II and 10 μM Ang-(1-9) were determined by Western-blot. (E) Confocal images of indirect immunofluorescence and detection of autophagosome's LC3 dot pattern (green), and nuclei staining (blue, Hoescht 33342) in cardiomyocytes cultured by 24 h with 10 nM Ang-II and 10 μM Ang-(1-9). Scale bar = 2 µm. Graphs represent the average ± SEM from at least n = 3 of protein levels. Data were relativized to GAPDH levels and normalized by NT condition. *p < 0.05 condition vs. 0 h or NT; **p < 0.01 condition vs. 0 h or NT, ***p < 0.001 condition vs. 0 h or NT, #p < 0,05 and & p < 0.01…

• 

  Figure 3. Ang-(1.9) activates the AT2R/Akt/ERK signaling pathway and modifies the cardiomyocyte autophagic tone through AT2R activation. Cardiomyocytes were cultured from 10 to 60 min with 10 μM Ang-(1-9) to determine the phosphorylation of (A) AKT and ERK1/2 through Western blot. (B) Graphs represent the average ± SEM from n = 3 of protein levels. Data were relativized to total AKT and ERK1/2, respectively, and normalized by basal conditions. *p < 0.05 condition vs. 0 h; **p < 0.01 condition vs. 0 h, ***p < 0.001 condition vs. 0 h. One-way ANOVA and Dunnet's post-test. Cells were treated for 30 min with the AT2R antagonist, PD123319 (2 μM) before stimulation with 10 μM Ang-(1-9) for 15 min and (C) p-AKT, and (D) p-ERK1/2 were determined through Western blot. Graphs represent the average ± SEM from n = 3 of protein levels. Data were relativized to total AKT, and total ERK1/2, respectively, and normalized by basal conditions. *p < 0.05 Ang-(1-9) vs. basal; **p < 0.01 Ang-(1-9) vs. basal, ****p < 0.0001 Ang-(1-9) vs. basal, #p < 0.05 PD vs. Control. Two-way ANOVA with Tukey's post-test. (E) Confocal images of indirect immunofluorescence and detection of autophagosome's LC3 dot pattern…

• 

  Figure 4. Angiotensin-(1-9) induces BCN1 expression and inhibitory phosphorylations. (A) Cardiomyocytes were cultured for 3 and 6 h with 10 μM Ang-(1-9) and levels of BCN1 were detected through Western blot. The graph represents the average ± SEM of n = 3. (B) Cells were cultured by 24 h with 10 nM Ang-II and 10 μM Ang-(1-9) (the last 6 h) and BCN1 protein levels were detected through Western blot. The graph represents the average ± SEM from at least n = 3 of protein levels. Data were relativized to GAPDH levels and normalized by NT condition. (C) Cells were treated for 5–60 min with 10 μM Ang-(1-9) and time-dependent phosphorylation of S234 and S295-BCN1 residues were detected by Western blot. The graph represents the average ± SEM of n = 4. Cardiomyocytes were treated with 2 μM PD123319 (PD), 5 μM Akti-1/2 (Akti) or 10 μM UO126 (UO) for 30 min before to 10 μM Ang-(1-9) by 10 min to detect through Western-blot the levels of phospho-S234-BCN1 (D) and phospho-S295-BCN1 (E) Graphs represent the average ± SEM of n = 4. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001 condition vs. 0 h or NT. One-way ANOVA and Dunnet's post-test.

• 

  Figure 5. Summary scheme. Ang-(1-9) triggers Akt activation via AT2R and leads the phosphorylation of BCN1 at S234 and S295 which in turn reduces the autophagy levels.