Neuregulin-4 attenuates diabetic cardiomyopathy by regulating autophagy via the AMPK/mTOR signalling pathway

Abstract

Background: Diabetic cardiomyopathy is characterized by left ventricle dysfunction, cardiomyocyte apoptosis, and interstitial fibrosis and is a serious complication of diabetes mellitus (DM). Autophagy is a mechanism that is essential for maintaining normal heart morphology and function, and its dysregulation can produce pathological effects on diabetic hearts. Neuregulin-4 (Nrg4) is an adipokine that exerts protective effects against metabolic disorders and insulin resistance. The aim of this study was to explore whether Nrg4 could ameliorate DM-induced myocardial injury by regulating autophagy.

Methods: Four weeks after the establishment of a model of type 1 diabetes in mice, the mice received Nrg4 treatment (with or without an autophagy inhibitor) for another 4 weeks. The cardiac functions, histological structures and cardiomyocyte apoptosis were investigated. Autophagy-related protein levels along with related signalling pathways that regulate autophagy were evaluated. In addition, the effects of Nrg4 on autophagy were also determined in cultured primary cardiomyocytes.

Results: Nrg4 alleviated myocardial injury both in vivo and in vitro. The autophagy level was decreased in type 1 diabetic mice, and Nrg4 intervention reactivated autophagy. Furthermore, Nrg4 intervention was found to activate autophagy via the AMPK/mTOR signalling pathway. Moreover, when autophagy was suppressed or the AMPK/mTOR pathway was inhibited, the beneficial effects of Nrg4 were diminished.

Conclusion: Nrg4 intervention attenuated diabetic cardiomyopathy by promoting autophagy in type 1 diabetic mice. Additionally, Nrg4 induced autophagy via the AMPK/mTOR signalling pathway.

Keywords: Autophagy; Diabetic cardiomyopathy; Neuregulin-4; Signalling pathway.

Fig. 1

Nrg4 alleviates cardiac hypertrophy and cardiac dysfunction. a Representative cardiac and tibial specimens of each group. b Cross-sectional view of cardiac specimens from each group. c Ratio of heart weight to tibia length (HW/TL). d Representative echocardiographic images. e Left ventricular ejection fraction (LVEF). f Left ventricular fraction shortening (LVFS). g Left ventricular end diastolic volume (LVEDV). Data are presented as the mean ± SD (n = 6 per group). Significant differences are depicted with ^*P < 0.05

Fig. 2

Nrg4 alleviates cardiac fibrosis and cardiomyocyte apoptosis. a Left ventricle stained with HE (original magnification ✕ 200). b Interstitial fibrosis of the left ventricles by Masson staining. The collagen fibres are stained blue (with arrows, original magnification ✕ 200). c Comparison of the collagen volume fraction (CVF) in each group. d–g Western blot and quantitative analysis of apoptosis-related proteins (Bax, Caspase-3 and Bcl-2). h Representative immunofluorescent images with TUNEL (green) and DAPI (blue) staining (original magnification ✕ 200). i Comparison of the apoptosis index in each group. Data are presented as the mean ± SD (n = 6 per group). Significant differences are depicted with ^*P < 0.05

Fig. 3

Nrg4 upregulates the suppressed autophagy in type 1 DM, and 3-MA attenuates Nrg4-upregulated autophagy. a, b Western blot and quantitative analysis of autophagy-related proteins (Beclin1, LC3-II/I and P62) in vivo. c Representative TEM images depicting autophagosomes in vitro, with arrows indicating autophagosomes. d, e Western blot and quantitative analysis of autophagy-related proteins in step 2 of the animal experiment. Data are presented as the mean ± SD (n = 6 per group). Significant differences are depicted with ^*P < 0.05

Fig. 4

3-MA attenuates the protective effect of Nrg4 on diabetic cardiomyopathy. a Representative immunofluorescent images with TUNEL (green) and DAPI (blue) staining (original magnification ✕ 200). b Comparison of the apoptosis index in each group. c Stained with HE (original magnification ✕ 200). d Stained with Masson staining (original magnification ✕ 200). e Comparison of CVF in each group. f Representative echocardiographic images. g Quantitative analysis of LVEF, LVFS and LVEDV. Data are presented as the mean ± SD (n = 6 per group). Significant differences are depicted with ^*P < 0.05

Fig. 5

Nrg4 activates autophagy via the AMPK/mTOR signalling pathway. a–c Western blot and quantitative analysis of the expression of p-AMPK and p-mTOR in vivo. (n = 6 per group). d–g Western blot and quantitative analysis of p-AMPK, p-mTOR and p-ULK1 in vitro (n = 3 independent experiments). h–k Western blot and quantitative analysis of autophagy-related proteins in vitro (n = 3 independent experiments). All data are presented as the mean ± SD. Significant differences are depicted with ^*P < 0.05

Fig. 6

Representative TEM images depicting autophagosomes in the vitro experiment (with arrows indicating autophagosomes)

Fig. 7

Compound C attenuates the protective effect of Nrg4 on cardiomyocyte apoptosis. a Representative immunofluorescent images with TUNEL in vitro (original magnification × 200). b Apoptosis indexes of different groups in vitro. Data are presented as the mean ± SD (n = 3 independent experiments). Significant differences are depicted with ^*P < 0.05

Fig. 8

Schematic diagram of the mechanisms of autophagy induced by Nrg4 intervention