Diabetes Metab J.  2021 Jul;45(4):594-605. 10.4093/dmj.2020.0049.

Role of Autophagy in Granulocyte-Colony Stimulating Factor Induced Anti-Apoptotic Effects in Diabetic Cardiomyopathy

• Shen GY
• Shin JH
• Song YS
• Joo HW
• Park IH
• Seong JH
• Shin NK
• Lee AH
• Cho YJ
• Lee Y
• Lim YH
• Kim H
• Kim KS

• KMID: 2518901
• DOI: http://doi.org/10.4093/dmj.2020.0049

Abstract

Background
We previously, reported that granulocyte-colony stimulating factor (G-CSF) reduces cardiomyocyte apoptosis in diabetic cardiomyopathy. However, the underlying mechanisms are not yet fully understood. Therefore, we investigated whether the mechanisms underlying of the anti-apoptotic effects of G-CSF were associated with autophagy using a rat model of diabetic cardiomyopathy.
Methods
Diabetic cardiomyopathy was induced in rats through a high-fat diet combined with low-dose streptozotocin and the rats were then treated with G-CSF for 5 days. Rat H9c2 cardiac cells were cultured under high glucose conditions as an in vitro model of diabetic cardiomyopathy. The extent of apoptosis and protein levels related to autophagy (Beclin-1, microtubule-binding protein light chain 3 [LC3]-II/LC3-I ratio, and P62) were determined for both models. Autophagy determination was performed using an Autophagy Detection kit.
Results
G-CSF significantly reduced cardiomyocyte apoptosis in the diabetic myocardium in vivo and led to an increase in Beclin-1 level and the LC3-II/LC3-I ratio, and decreased P62 level. Similarly, G-CSF suppressed apoptosis, increased Beclin-1 level and LC3-II/LC3-I ratio, and decreased P62 level in high glucose-induced H9c2 cardiac cells in vitro. These effects of G-CSF were abrogated by 3-methyladenine, an autophagy inhibitor. In addition, G-CSF significantly increased autophagic flux in vitro.
Conclusion
Our results suggest that the anti-apoptotic effect of G-CSF might be significantly associated with the up-regulation of autophagy in diabetic cardiomyopathy.

Keyword

Apoptosis; Autophagy; Diabetic cardiomyopathies; Granulocyte colony-stimulating factor; Myocytes, cardiac

Figure

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  Fig. 1. Scheme of the animal experiment. Diabetes was induced in rats by feeding for 7 weeks with a high-fat diet and low-dose streptozotocin (30 mg/kg) injection. Rat were then randomized for treatment with granulocyte-colony stimulating factor (G-CSF) or saline administrated intraperitoneally, for 5 days. Body weight, biochemical analysis, and echocardiography were performed both pre- and post-treatment. At 18 weeks of age, all rats were euthanized for histology and protein analysis. SD, Sprague-Dawley.

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  Fig. 2. Effect of granulocyte-colony stimulating factor (G-CSF) on myocardial apoptosis in the diabetic myocardium. (A) Representative images of terminal deoxynucleotidyl transferase (TDT)–mediated dUTP–biotin nick end–labeling (TUNEL) assay staining of myocardium for each group 4 weeks after treatment (magnification ×400). Apoptotic nuclei are stained brown and non-apoptotic nuclei are stained blue on TUNEL assay staining. (B) Quantitative analysis of apoptotic cells in the myocardium of each group. (C) Level of B-cell lymphoma 2 (Bcl-2) protein in cardiac tissue was detected by Western blotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (D) Quantitative Western blot analysis of Bcl-2. The expression level was normalized by comparison with GAPDH expression. Protein levels are expressed as mean±standard deviation. Histology data are expressed as mean±standard error. aP<0.05 vs. normal group, bP<0.05 vs. saline group (n=6–8 per group).

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  Fig. 3. Effect of granulocyte-colony stimulating factor (G-CSF) on autophagy in the diabetic myocardium. (A) Representative images showing the levels of autophagy-related proteins Beclin-1, the microtubule-binding protein light chain 3 (LC3)-II/LC3-I ratio, and P62 in the diabetic myocardium measured by Western blot at 18 weeks of age (4 weeks after G-CSF and saline treatment). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B, C, D) Quantitative Western blot analysis of Beclin-1, the LC3-II/LC3-I ratio, and P62. Protein levels were normalized by comparison with GAPDH expression. All data are expressed as mean±standard deviation. aP<0.05 vs. normal group, bP<0.05 vs. saline group (n=6–8 per group).

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  Fig. 4. Effect of granulocyte-colony stimulating factor (G-CSF) on apoptosis in high glucose-induced H9c2 cardiac cells. (A) Dot plots displaying the stages of apoptotic death of H9c2 cardiac cells: Annexin−/PI− (Q3), viable cells; Annexin+/PI− (Q4), cells undergoing apoptosis; Annexin+/PI+ (Q2), cells in end-stage apoptosis or that are already dead; Annexin−/PI+ (Q1), cells that are in necrosis. (a) H9c2 cardiac cells cultured in low glucose media; (b) H9c2 cardiac cells cultured in high glucose media; (c) H9c2 cardiac cells cultured in high glucose media containing G-CSF (3 μg/mL); (d) H9c2 cardiac cells cultured in high glucose media containing G-CSF (3 μg/mL) and 3-methyladenine (3-MA; 5 mM). (B) Quantitative analysis of apoptotic cells (Q2+Q4). All data are expressed as mean±standard deviation. FITC, fluorescein isothiocyanate. aP<0.05 vs. H9c2 cardiac cells cultured in low glucose media, bP<0.05 vs. H9c2 cardiac cells cultured in high glucose media, cP<0.05 vs. H9c2 cardiac cells cultured in high glucose media containing G-CSF (n=5 per group).

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  Fig. 5. Effect of granulocyte-colony stimulating factor (G-CSF) on autophagy in high glucose-induced H9c2 cardiac cells. (A) Representative images of levels of autophagy-related proteins Beclin-1, the microtubule-binding protein light chain 3 (LC3)-II/LC3-I ratio, and P62 in H9c2 cardiac cells measured by Western blot. (B, C, D) Quantitative Western blot analysis of Beclin-1, the LC3-II/LC3-I ratio, and P62. Protein levels were normalized by comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. GAPDH was used as a loading control. All data are expressed as mean±standard deviation. 3-MA, 3-methyladenine. aP<0.05 vs. H9c2 cardiac cells cultured in low glucose media, bP<0.05 vs. H9c2 cardiac cells cultured in high glucose media, cP<0.05 vs. H9c2 cardiac cells cultured in high glucose media containing G-CSF (n=5 per group).

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  Fig. 6. Effect of granulocyte-colony stimulating factor (G-CSF) on autophagic flux in high glucose-induced H9c2 cardiac cells. Autophagic flux evaluated high glucose-induced H9c2 cardiac cells by flow cytometry and fluorescence microscopy. Representative histogram (A) and bar graph (B), where fluorescence increase of autophagy green indicates autophagic flux increase. (C) Autophagic flux evaluated by fluorescence microscopy (magnification ×400), photos are representative of four to five independent experiments. Arrows indicate autophagic vesicles. The inserts in (C) show higher magnification. Rapamycin was used as a positive control of autophagy. All data are expressed as mean±standard deviation. Normal, normal condition group. aP<0.05 vs. H9c2 cardiac cells cultured in normal condition, bP<0.05 vs. H9c2 cardiac cells cultured in high glucose media containing G-CSF (n=4–5 per group).

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