The CREG1-FBXO27-LAMP2 axis alleviates diabetic cardiomyopathy by promoting autophagy in cardiomyocytes

Abstract

Autophagy plays an important role in the development of diabetic cardiomyopathy. Cellular repressor of E1A-stimulated genes 1 (CREG1) is an important myocardial protective factor. The aim of this study was to investigate the effects and mechanisms of CREG1 in diabetic cardiomyopathy. Male C57BL/6 J mice, Creg1 transgenic mice and cardiac-specific knockout mice were used to establish a type 2 diabetes model. Small animal ultrasound, Masson's staining and western blotting were used to evaluate cardiac function, myocardial fibrosis and autophagy. Neonatal mouse cardiomyocytes (NMCMs) were stimulated with palmitate, and the effects of CREG1 on NMCMs autophagy were examined. CREG1 deficiency exacerbated cardiac dysfunction, cardiac hypertrophy and fibrosis in mice with diabetic cardiomyopathy, which was accompanied by exacerbated autophagy dysfunction. CREG1 overexpression improved cardiac function and ameliorated cardiac hypertrophy and fibrosis in diabetic cardiomyopathy by improving autophagy. CREG1 protein expression was decreased in palmitate-induced NMCMs. CREG1 knockdown exacerbated cardiomyocyte hypertrophy and inhibited autophagy. CREG1 overexpression inhibited cardiomyocyte hypertrophy and improved autophagy. LAMP2 overexpression reversed the effect of CREG1 knockdown on palmitate-induced inhibition of cardiomyocyte autophagy. CREG1 inhibited LAMP2 protein degradation by inhibiting the protein expression of F-box protein 27 (FBXO27). Our findings indicate new roles of CREG1 in the development of diabetic cardiomyopathy.

Fig. 1. CREG1 deficiency exacerbates cardiac hypertrophy and autophagy dysfunction in type 2 diabetic mice.

a The E/A ratio of Creg1^fl/fl and Creg1-CKO mice after type 2 DM was established (24 weeks, n = 6). b, c EF% and FS% in Creg1^fl/fl and Creg1-CKO mice after type 2 DM was established (24 weeks, n = 6). d–f HE staining and Masson’s trichrome staining of control and DM mice in the 24th week (n = 3). g, h WGA staining of the myocardium of control and DM mice in the 24th week (n = 4). i Effects of CREG1 deficiency on the mRNA levels of hypertrophic (Anp and Myh7) and fibrotic (Tgfβ) markers in the myocardium of control and DM mice in the 24th week. j, k Expression of CREG1 and autophagic and lysosomal proteins in the myocardium of Creg1^fl/fl and Creg1-CKO mice (n = 3). Creg1^fl/fl: littermate control, Creg1-CKO: Creg1 cardiac knockout mice, DM: diabetic model, *p < 0.05, **p < 0.01 vs. Creg1^fl/fl; ^##p < 0.01 vs. Creg1-CKO; ^&p < 0.05, ^&&p < 0.01 vs. Creg1^fl/fl-DM.

Fig. 2. CREG1 overexpression ameliorates cardiac hypertrophy and autophagy dysfunction in type 2 diabetic mice.

a The E/A ratio in WT and Creg1-TG mice after type 2 DM was established (24 weeks, n = 7). b, c EF% and FS% in WT and Creg1-TG mice after type 2 DM was established (24 weeks, n = 7). d–f HE staining and Masson’s trichrome staining of control and DM mice in the 24th week (n = 3). g, h WGA staining of the myocardium of control and DM mice in the 24th week (n = 4). i Effects of CREG1 overexpression on the mRNA levels of hypertrophic (Anp and Myh7) and fibrotic (Tgfβ) marker genes in the myocardium of control and DM mice in the 24th week. j, k Expression of CREG1, autophagy-related proteins and lysosome-related proteins in the myocardium of WT and Creg1-TG mice after DM was established (n = 3). WT: littermate control, Creg1-TG: Creg1 transgenic mice, DM: diabetic model. *p < 0.05, **p < 0.01 vs. WT; ^#p < 0.05, ^##p < 0.01 vs. Creg1-TG; ^&p < 0.05, ^&&p < 0.01 vs. WT-DM.

Fig. 3. CREG1 deficiency exacerbates palmitate-induced autophagy dysfunction in cardiomyocytes.

a The mRNA expression of Creg1 in NMCMs after PA stimulation (n = 3). b, c The protein expression of CREG1 in NMCMs after PA stimulation (n = 4). d Effects of CREG1 knockdown on the mRNA expression of Anp and Myh7 (n = 3). e, f F-actin staining in NMCMs with CREG1 knockdown and PA stimulation (n = 5). g, h Effects of CREG1 knockdown on the expression of autophagy-related proteins and lysosome-related proteins (n = 4). i, j Effects of CREG1 knockdown on autophagic flux in NMCMs (n = 3). NMCMs: neonatal mouse cardiomyocytes, PA: palmitate. **p < 0.01 vs. control or si-control, ^#p < 0.05, ^##p < 0.01 vs. si-Creg1; ^&p < 0.05, ^&&p < 0.01 vs. si-control+PA.

Fig. 4. CREG1 overexpression ameliorates palmitate-induced autophagy dysfunction in cardiomyocytes.

a The mRNA expression of Anp and Myh7 in NMCMs with CREG1 overexpression and PA stimulation (n = 3). b, c F-actin staining in NMCMs with CREG1 overexpression and PA stimulation (n = 5). d, e Effects of CREG1 overexpression on the expression of autophagy-related proteins and lysosomal-related proteins (n = 4). f, g Effects of CREG1 overexpression on autophagosomes and autophagolysosomes in NMCMs (n = 3). NMCMs: neonatal mouse cardiomyocytes, PA: palmitate. **p < 0.01 vs. adcon; ^#p < 0.05, ^##p < 0.01 vs. adCREG1; ^&p < 0.05, ^&&p < 0.01 vs. adcon+PA.

Fig. 5. CREG1 overexpression ameliorates cardiomyocyte hypertrophy by activating cardiomyocyte autophagy.

a, b Effects of CREG1 overexpression on the expression of autophagy-related proteins in CQ-induced NMCMs (n = 3). c, d Effects of CREG1 overexpression on autophagosomes and autophagolysosomes in CQ-induced NMCMs (n = 3). e The mRNA expression of Anp and Myh7 in CREG1-overexpressing NMCMs following CQ stimulation (n = 3). f, g F-actin staining in CREG1-overexpressing NMCMs following CQ stimulation (n = 5). NMCMs: neonatal mouse cardiomyocytes, PA: palmitate (400 μM, 24 h), CQ: chloroquine (20 μM, 24 h). *p < 0.05, **p < 0.01 vs. adcon+PA; ^#p < 0.05, ^##p < 0.01 vs. adCREG1+PA; ^&p < 0.05, ^&&p < 0.01 vs. adcon+CQ+PA.

Fig. 6. CREG1 deficiency exacerbates autophagy dysfunction in cardiomyocytes by inhibiting LAMP2 protein expression.

a Immunoprecipitation of CREG1 with LAMP2 in H9C2 cells. b Immunofluorescence staining of CREG1 and LAMP2 in NMCMs. c Real-time PCR analysis of Lamp2 mRNA expression in CREG1-overexpressing NMCMs. d, e Western blotting analysis of LAMP2 protein expression in CREG1-overexpressing NMCMs. f Real-time PCR analysis of Creg1 mRNA expression in LAMP2-overexpressing NMCMs. g, h Western blotting analysis of CREG1 protein expression in LAMP2-overexpressing NMCMs. i, j Effects of CREG1 knockdown and LAMP2 overexpression on the expression of autophagy-related proteins and lysosomal-related proteins in NMCMs, as determined by western blotting. k, l Effects of CREG1 knockdown and LAMP2 overexpression on autophagic flux in NMCMs. NMCMs: neonatal mouse cardiomyocytes, PA: palmitate. *p < 0.05, **p < 0.01 vs. adcon or si-control; ^#p < 0.05, ^##p < 0.01 vs. si-control+PA; ^&p < 0.05, ^&&p < 0.01 vs. si-Creg1 + PA, n = 3.

Fig. 7. CREG1 inhibits the degradation of LAMP2 in a FBXO27-dependent manner.

a, b Western blotting analysis of LAMP2 or CREG1 protein expression in NMCMs following stimulation with CQ or MG132. c Real-time PCR analysis of the mRNA expression of Fbxo6 and Fbxo27 in CREG1-overexpressing NMCMs. d, e Western blotting analysis of the protein expression of FBXO6 and FBXO27 in CREG1-overexpressing NMCMs. f Real-time PCR analysis of the mRNA expression of Creg1 and Lamp2 in FBXO27-overexpressing NMCMs. g, h Western blotting analysis of the protein expression of CREG1 and LAMP2 in FBXO27-overexpressing NMCMs. i, j Effects of CREG1 and FBXO27 overexpression on the expression of autophagy-related proteins in NMCMs, as determined by western blotting. k Schematic illustration of the proposed mechanism of CREG1 in diabetic cardiomyopathy. NMCMs: neonatal mouse cardiomyocytes, CQ: chloroquine (20 μM, 24 h), PA: palmitate (400 μM, 24 h). **p < 0.01 vs. si-control or adcon or control; ^##p < 0.01 vs. si-Creg1 or PA; ^&p < 0.05, ^&&p < 0.01 vs. si-Creg1 + CQ or FBXO27 + PA, n = 3.