Lab Anim Res.  2012 Jun;28(2):99-107. 10.5625/lar.2012.28.2.99.

Autophagy contributes to retardation of cardiac growth in diabetic rats

• Lee Y
• Hong Y
• Lee SR
• Chang KT
• Hong Y

• KMID: 1436703
• DOI: http://doi.org/10.5625/lar.2012.28.2.99

Abstract

Diabetes mellitus is a major predictor of heart failure, although the mechanisms by which the disease causes cardiomyopathy are not well understood. The purpose of this study was to determine whether prolonged exposure of cardiomyocytes to high glucose concentrations induces autophagy and contributes to cardiomyopathy. Interestingly, there were no differences in the autophagic activation produced by different glucose concentrations. However, cell viability was decreased by high glucose. In the diabetic rats, we found a higher level of microtubule-associated protein light chain 3 (LC3) expression and a reduction in the size of the left ventricle (LV) (P<0.05) caused by growth retardation, suggesting activated autophagy. Our in vitro findings indicate that hyperglycemic oxidative stress induces autophagy, and our in vivo studies reveal that autophagy is involved in the progression of pathophysiological remodeling of the heart. Taken together, the studies suggest that autophagy may play a role in the pathogenesis of juvenile diabetic cardiomyopathy.

Keyword

Autophagy; cardiomyopathy; diabetes; growth retardation; hyperglycemia

MeSH Terms

Animals
Autophagy
Cardiomyopathies
Cell Survival
Diabetes Mellitus
Diabetic Cardiomyopathies
Glucose
Heart
Heart Failure
Heart Ventricles
Hyperglycemia
Light
Myocytes, Cardiac
Oxidative Stress
Rats
Glucose

Figure

• 

  Figure 1 Glucose starvation strongly induces cell-size reduction and autophagy. A, Representative inverted microscopy images after 12 h of glucose starvation. Magnification ×100. Scale bar=40 µm. B, Immunoblot analysis of microtubule-associated protein-1 light chain-3 (LC3)-I and LC3-II. Cardiac myocytes were incubated with complete media (control) or treated with glucose-free [Glu(-)] medium for 12 h. In A and B, the results represent data from three independent experiments. **P<0.01

• 

  Figure 2 High glucose media suppresses proliferation of cardiomyocytes, and induces cell death with accompanied by autophagy. A, Representative light microscopy images of H9c2 cells. Cells were treated with complete media as a control (Con) or with glucose-free [Glu(-)], 33 mM glucose [Glu(2×)], or 66 mM glucose [Glu(4×)] media for 36 hr and 72 hr. Scale bar = 100 um. B, Time course of cell viability after media exchange, estimated by trypan blue assay. Values are mean from three independent experiments. **P<0.01 vs. Con. C, Immunoblot analysis of LC3-I and LC3-II. Cells were cultured in complete media as a control (Con), glucose-free media [Glu(-)], 33 mM glucose [Glu(2×)], or 66 mM glucose [Glu(4×)] for 72 hr, and quantitative analysis of relative LC3-II expression to compare the level of autophagy between groups. *P<0.05 vs. Con, **P<0.01 vs. Con. D, Time course of LC3 expression by immunoblot analysis after 66 mM glucose [Glu(4×)] treatment, and quantitative analysis of relative LC3-II expression to compare the level of autophagy between time points in Glu(4×) group. *P<0.05 vs. Con

• 

  Figure 3 General features of diabetic rats. A, Body weight of STZ-induced diabetic vs. control rats. *P<0.01 B, Blood pressure of STZ-induced diabetic vs. control rats. C, Blood glucose level of STZ-induced diabetic vs. control rats. *P<0.01 D, Measurement of tibia length in control and diabetic rats after 14 and 28 days of STZ treatment. In comparison to the untreated control, the diabetic rats show suppressed bone growth. *P<0.05.

• 

  Figure 4 The size of the left ventricle is reduced in diabetic rats with cardiac muscle atropy. After 4 weeks, three control and three diabetic animals were sacrificed. A, Representative morphological images of wet left ventricular tissue showing that early-onset diabetes causes the suppression of heart development. (a, b) Representative cross sections of the heart showing the reduced thickness of the left ventricular wall. Scale bar=0.5 cm. (c, d) B, The effects of diabetes on the left ventricle mass to body weight (LV/BW) index (upper panel), and the ratio of the left ventricle mass to tibia length was also decreased (lower panel). *P<0.05 C, Hematoxylin-eosin (H&E) stained cross-sections of rat heart muscles after 28 days of streptozotocin treatment (DM) or vehicle treatment (Con). Distribution of nuclei was further confirmed by propidium iodide (PI) staining. Magnification ×400, scale bar=50 µm. D, Comparative analysis of the diameter of muscle fibers between groups. *P<0.05 vs. Con

• 

  Figure 5 Diabetes-induced myocardial autophagy. A, Immunohistochemistry using antibody to microtubule-associated protein-1 light chain-3 (LC3). Increased abundance of the autophagosomal marker, LC3, in diabetic rats compared to control rats. Magnification ×100, scale bar=100 µm. B, Representative immunoblots probed for LC3, beclin 1, and caspase 3 in ventricular lysates, as indicated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.

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