Autophagic adaptations in diabetic cardiomyopathy differ between type 1 and type 2 diabetes

Abstract

Little is known about the association between autophagy and diabetic cardiomyopathy. Also unknown are possible distinguishing features of cardiac autophagy in type 1 and type 2 diabetes. In hearts from streptozotocin-induced type 1 diabetic mice, diastolic function was impaired, though autophagic activity was significantly increased, as evidenced by increases in microtubule-associated protein 1 light chain 3/LC3 and LC3-II/-I ratios, SQSTM1/p62 (sequestosome 1) and CTSD (cathepsin D), and by the abundance of autophagic vacuoles and lysosomes detected electron-microscopically. AMP-activated protein kinase (AMPK) was activated and ATP content was reduced in type 1 diabetic hearts. Treatment with chloroquine, an autophagy inhibitor, worsened cardiac performance in type 1 diabetes. In addition, hearts from db/db type 2 diabetic model mice exhibited poorer diastolic function than control hearts from db/+ mice. However, levels of LC3-II, SQSTM1 and phosphorylated MTOR (mechanistic target of rapamycin) were increased, but CTSD was decreased and very few lysosomes were detected ultrastructurally, despite the abundance of autophagic vacuoles. AMPK activity was suppressed and ATP content was reduced in type 2 diabetic hearts. These findings suggest the autophagic process is suppressed at the final digestion step in type 2 diabetic hearts. Resveratrol, an autophagy enhancer, mitigated diastolic dysfunction, while chloroquine had the opposite effects in type 2 diabetic hearts. Autophagy in the heart is enhanced in type 1 diabetes, but is suppressed in type 2 diabetes. This difference provides important insight into the pathophysiology of diabetic cardiomyopathy, which is essential for the development of new treatment strategies.

Keywords: AMP-activated protein kinase; AMPK, AMP-activated protein kinase; CTSD, cathepsin D; DM, diabetes mellitus; GFP, green fluorescent protein; HBA1c, glycated hemoglobin α 1; LV, left ventricular; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MTOR, mechanistic target of rapamycin; Mn-SOD, superoxide dismutase 2, mitochondrial; SIRT1, sirtuin 1; SQSTM1/p62, sequestosome 1; STZ, streptozotocin; autophagy; cardiomyopathy; chloroquine; diabetes mellitus; insulin; resveratrol; type 1 diabetes; type 2 diabetes; ultrastructure.

Figure 1.

Diabetic profile and cardiac function of type 1 diabetic mice. (A) Diabetic profile in nondiabetic (Sham) and streptozotocin (STZ)-induced type 1 diabetic mice (n = 12 each). *P < 0.05 vs. nondiabetic control group. (B) Cardiac geometry and function assessed by echocardiography. Cq, chloroquine; pre- and post-treatment with Cq; EF, LV ejection fraction; LVDd, LV diastolic diameter; HR, heart rate. *P < 0.05 vs. pretreatment. (C) Cardiac function assessed by cardiac catheterization. LVSP, LV peak systolic pressure; LVEDP, LV end-diastolic pressure; +dP/dt and –dP/dt, maximal and minimal first derivative of LV pressure. *P < 0.05 vs. saline-treated sham; ^†P < 0.05 vs. saline-treated STZ.

Figure 2.

Diabetic profile and cardiac function of type 2 diabetic mice. (A) Diabetic profile of db/+ nondiabetic and db/db type 2 diabetic mice (n = 12 each). *P < 0.05 vs. the db/+ nondiabetic control group; ^†P < 0.05 vs. db/db diabetic group. (B) Cardiac geometry and function assessed by echocardiography. Cq, chloroquine; RSV, resveratrol; pre- and post-treatment with Cq or RSV; EF, LV ejection fraction; LVDd, LV diastolic diameter; HR, heart rate. *P < 0.05 vs. pretreatment. (C) Cardiac function assessed by cardiac catheterization. LVSP, LV peak systolic pressure; LVEDP, LV end-diastolic pressure; +dP/dt and –dP/dt, maximal and minimal first derivative of LV pressure. *P < 0.05 vs. saline-treated db/+; ^†P < 0.05 vs. saline-treated db/db.

Figure 3.

Autophagic profile in type 1 diabetic mice. (A) Accumulation of autophagic vacuoles. Immunofluorescent labeling of GFP-LC3 (green dots) and MB/myoglobin (red) within cardiomyocytes. Scale bars: 20 μm. The graph shows the numbers of GFP-LC3 dots per high-power field (HPF) (600×) (n = 6 each). (B) Western blotting with densitometric analysis of the autophagy-related proteins LC3 and SQSTM1 in hearts (n = 6 each). (C) Lysosomal activity in nondiabetic and STZ-induced diabetic hearts assessed by immunohistochemistry for CTSD and western blotting with densitometric analysis of CTSD (n = 6). Bars in photographs: 20 μm. *P < 0.05 vs. the nondiabetic control group; ^†P < 0.05 vs. STZ-treated diabetic group. STZ, streptozotocin; Cq, chloroquine.

Figure 4.

Electron micrographs of autophagic vacuoles in cardiomyocytes. (A) Nondiabetic control heart. ((B) and B') STZ-treated diabetic heart; B' is an enlargement of the boxed area in B. (C) Nondiabetic heart with chloroquine intervention. (D) STZ-treated diabetic heart with chloroquine intervention. Arrowheads and arrows indicate lysosomes (electron-dense spherical bodies) and autophagic vacuoles, respectively. Scale bars: 1 μm. Nucl, nucleus; Mt, mitochondria. (E) Quantification of autophagic vacuoles and lysosomes within cardiomyocytes under an electron microscope. Y-axis indicates the number of autophagic vacuoles (left panel) or lysosomes (right panel) per printed field (289 μm²) within a cardiomyocyte. *P < 0.05 vs. the nondiabetic control group; ^†P < 0.05 vs. STZ-treated diabetic group.

Figure 5.

Assessment of myocardial energy status and autophagy-related signals. (A) Western blotting with densitometric analysis of AMPK, p-AMPK, MTOR and p-MTOR (n = 6 each). (B) Myocardial ATP content in nondiabetic and STZ-induced diabetic hearts (n = 6 each). *P < 0.05 vs. the nondiabetic control group; ^†P < 0.05 vs. STZ-treated diabetic group. STZ, streptozotocin; Cq, chloroquine.

Figure 6.

Autophagic profile of type 2 diabetic mice. (A) Accumulation of autophagic vacuoles. Immunofluorescent labeling of LC3 (green) and MB/myoglobin (red) within cardiomyocytes. Nuclei were stained blue with Hoechst 33342. Scale bars: 20 μm. The graph shows the numbers of LC3-positive (green) dots per high-power field (HPF) (600×) (n = 6 each). (B) Western blotting with densitometric analysis of the autophagy-related proteins LC3 and SQSTM1 in hearts. Graphs show the intensity of each band in arbitrary units and the LC3-II/LC3-I ratios (n = 6 each). (C) Lysosomal activity in db/+ nondiabetic and db/db diabetic hearts assessed by immunohistochemistry for CTSD and western blotting with densitometric analysis of CTSD (n = 6 each). Scale bar in micrographs: 20 μm. *P < 0.05 vs. the db/+ nondiabetic control group; ^†P < 0.05 vs. db/db diabetic group. S, saline; RSV, resveratrol; Cq, chloroquine.

Figure 7.

Electron micrographs of autophagic vacuoles in cardiomyocytes. (A) db/+ nondiabetic control heart. (B, C and C') db/db diabetic control hearts; C' is an enlargement of the boxed area in (C). (D and E) db/db hearts after resveratrol intervention. (F and G) db/db hearts after chloroquine intervention. Black arrowheads, white arrowheads and arrows indicate lysosomes (electron dense spherical bodies), lipids (homogenously electron-transparent light gray spherical bodies) and autophagic vacuoles, respectively. Scale bars: 1 μm.

Figure 8.

Quantification of autophagic vacuoles and lysosomes within cardiomyocytes under an electron microscope. Y-axis indicates the number of autophagic vacuoles (left panel) or lysosomes (right panel) per printed field (289 μm²) within a cardiomyocyte. *P < 0.05 vs. the db/+ nondiabetic control group; ^†P < 0.05 vs. db/db diabetic group.

Figure 9.

In vivo autophagic flux assay in db/+ nondiabetic and db/db type 2 diabetic mice. (A) Immunofluorescent labeling of LC3 (green) and myoglobin/MB (red) within cardiomyocytes. Nuclei were stained blue with Hoechst 33342. Scale bars: 20 μm. The graphs show the relative number of LC3 dots per HPF (600×) (n = 6 each). (B) Western blotting of LC3. The graphs show the relative levels of LC3-II expression determined using densitometry (n = 6 each). Sal, saline; Cq, chloroquine.

Figure 10.

Assessment of myocardial energy status and autophagy-related signals. (A) Western blotting with densitometric analysis of AMPK, p-AMPK, MTOR and p-MTOR. Graphs show the intensity of each band in arbitrary units (n = 6 each). (B) Myocardial ATP content (n = 6 each). *P < 0.05 vs. the db/+ nondiabetic control group; ^†P < 0.05 vs. db/db diabetic group. S, saline; RSV/R, resveratrol; Cq, Chloroquine.