Distinct cardiac energy metabolism and oxidative stress adaptations between obese and non-obese type 2 diabetes mellitus

Abstract

Background: Little is known about the pathophysiological diversity of myocardial injury in type 2 diabetes mellitus (T2DM), but analyzing these differences is important for the accurate diagnosis and precise treatment of diabetic cardiomyopathy. This study aimed to elucidate the key cardiac pathophysiological differences in myocardial injury between obese and non-obese T2DM from mice to humans. Methods: Obese and non-obese T2DM mouse models were successfully constructed and observed until systolic dysfunction occurred. Changes in cardiac structure, function, energy metabolism and oxidative stress were assessed by biochemical and pathological tests, echocardiography, free fatty acids (FFAs) uptake fluorescence imaging, transmission electron microscopy, etc. Key molecule changes were screened and verified by RNA sequencing, quantitative real-time polymerase chain reaction and western blotting. Further, 28 human heart samples of healthy population and T2DM patients were collected to observe the cardiac remodeling, energy metabolism and oxidative stress adaptations as measured by pathological and immunohistochemistry tests. Results: Obese T2DM mice exhibited more severe cardiac structure remodeling and earlier systolic dysfunction than non-obese mice. Moreover, obese T2DM mice exhibited severe and persistent myocardial lipotoxicity, mainly manifested by increased FFAs uptake, accumulation of lipid droplets and glycogen, accompanied by continuous activation of the peroxisome proliferator activated receptor alpha (PPARα) pathway and phosphorylated glycogen synthase kinase 3 beta (p-GSK-3β), and sustained inhibition of glucose transport protein 4 (GLUT4) and adipose triglyceride lipase (ATGL), whereas non-obese mice showed no myocardial lipotoxicity characteristics at systolic dysfunction stage, accompanied by the restored PPARα pathway and GLUT4, sustained inhibition of p-GSK-3β and activation of ATGL. Additionally, both obese and non-obese T2DM mice showed significant accumulation of reactive oxygen species (ROS) when systolic dysfunction occurred, but the NF-E2-related factor 2 (Nrf2) pathway was significantly activated in obese mice, while was significantly inhibited in non-obese mice. Furthermore, the key differences found in animals were reliably verified in human samples. Conclusion: Myocardial injury in obese and non-obese T2DM may represent two different types of complications. Obese T2DM individuals, compared to non-obese individuals, are more prone to develop cardiac systolic dysfunction due to severe and persistent myocardial lipotoxicity. Additionally, anti-oxidative dysfunction may be a key factor leading to myocardial injury in non-obese T2DM.

Figure 1

Technical roadmap of the study. Obese and non-obese T2DM mouse models were successfully constructed and the cardiac structure, function, metabolism (Met), oxidative stress (Os) and related molecular changes were systematically observed. Human heart samples of healthy population and T2DM patients were collected to observe the cardiac remodeling, energy metabolism and oxidative stress adaptations.

Figure 2

Cardiac structure of obese and non-obese T2DM mice. (A) Macroscopic pictures of hearts from obese T2DM mice in the 24th week of the disease course. (B) Heart weight and heart weight/tibia length ratio from obese T2DM mice in the 24th week of the disease course. (n = 6) (C) Fold change of mRNA expression levels of ANP and BNP in heart tissues from obese T2DM mice in the 24th week of the disease course. (n = 4-6) (D) HE staining (Scale bar = 100 μm), WGA staining (Scale bar = 30 μm), Masson staining (Scale bar = 50 μm) and CD31 immunofluorescence staining (Scale bar = 100 μm) of heart tissues from obese T2DM mice in the 24th week of the disease course. (E) Changes in cardiomyocyte size, collagen area ratio and microvessel density based on panel D. (n = 3) (F) Macroscopic pictures of hearts from non-obese T2DM mice in the 24th and 36th weeks of the disease course. (G) Heart weight and heart weight/tibia length ratio from non-obese T2DM mice in the 24th and 36th weeks of the disease course. (n = 6) (H) Fold change of mRNA expression levels of ANP and BNP in heart tissues from non-obese T2DM mice in the 24th and 36th weeks of the disease course. (n = 4-6) (I) HE staining (Scale bar = 100 μm), WGA staining (Scale bar = 30 μm), Masson staining (Scale bar = 50 μm) and CD31 immunofluorescence staining (Scale bar = 100 μm) of heart tissues from non-obese T2DM mice in the 24th and 36th weeks of the disease course. (J) Changes in cardiomyocyte size, collagen area ratio and microvessel density based on panel I. (n = 3) Data are expressed as mean ± SD. (*, p<0.05; **, p<0.01; ***, p<0.001).

Figure 3

Cardiac function of obese and non-obese T2DM mice. (A) Echocardiography of obese T2DM mice. (B) Data related to cardiac function of obese T2DM mice. (n = 6-10) (C) Echocardiography of non-obese T2DM mice. (D) Data related to cardiac function of non-obese T2DM mice. (n = 6-10) Data are expressed as mean ± SD. (*, p<0.05; **, p<0.01; ***, p < 0.001).

Figure 4

Metabolic situation in hearts of obese and non-obese T2DM mice. (A) Fluorescence imaging of cardiac FFAs uptake from obese T2DM mice in the 24th week of the disease course and mean photons per square millimeter calculated. (n = 3) (B) Oil red O staining (Scale bar = 100 μm), PAS staining (Scale bar = 50 μm; Black arrow indicates glycogen particles) and TEM pictures (Scale bar = 2 μm; Mt, mitochondria; Li, lipid droplet; Yellow arrow indicates glycogen particles) of heart tissues from obese T2DM mice in the 24th week of the disease course. (C) DHE staining of heart tissues from obese T2DM mice in the 24th week of the disease course (Scale bar = 50 μm) and the fluorescence intensity. (n = 3) (D) TUNEL staining of heart tissues from obese T2DM mice in the 24th week of the disease course (Scale bar = 20 μm) and the number of apoptotic cells. (n = 3) (E) Fluorescence imaging of cardiac FFAs uptake from non-obese T2DM mice in the 36th week of the disease course and mean photons per square millimeter calculated. (n = 3) (F) Oil red O staining (Scale bar = 100 μm), PAS staining (Scale bar = 50 μm; Black arrow indicates glycogen particles) and TEM pictures (Scale bar=2 μm; Mt, mitochondria; Li, lipid droplet; Yellow arrow indicates glycogen particles) of heart tissues from non-obese T2DM mice in the 24th and/or 36th week of the disease course. (G) DHE staining of heart tissues from non-obese T2DM mice in the 36th week of the disease course (Scale bar = 50 μm) and the fluorescence intensity. (n = 3) (H) TUNEL staining of heart tissues from non-obese T2DM mice in the 36th week of the disease course (Scale bar = 20 μm) and the number of apoptotic cells. (n = 3) Data are expressed as mean ± SD. (*, p <0.05; ***, p<0.001.).

Figure 5

RNA sequencing results. (A) Venn diagram of DEGs. (B) Common DEGs shared by the two models. (C) DEGs related to cardiomyopathy of the two models. (D) DEGs related to insulin resistance of the two models. (E) DEGs related to the metabolic pathway of the two models. (F) DEGs related to the PPAR pathway of the two models. (G) DEGs related to the PI3K/Akt pathway of the two models. (H) Q-PCR of genes related to the PPARα pathway in obese T2DM mice. (n = 4-6) (I) Q-PCR of genes related to the PPARα pathway in non-obese T2DM mice. (n = 3-4) Data are expressed as mean ± SD. (*, p<0.05; **, p<0.01; ***, p < 0.001).

Figure 6

Western blot results of proteins related to energy metabolism in heart tissues of obese T2DM mice. (A) Western blot images of proteins related to energy metabolism in heart tissues from obese T2DM mice. (B) Relative quantification based on the results of panel A. (n = 3) Data are expressed as mean ± SD. (*, p<0.05; **, p<0.01; ***, p < 0.001).

Figure 7

Western blot results of proteins related to energy metabolism in heart tissues of non-obese T2DM mice. (A) Western blot images of proteins related to energy metabolism in heart tissues from non-obese T2DM mice. (B) Relative quantification based on the results of panel C. (n = 3-6) Data are expressed as mean ± SD. (*, p<0.05; **, p<0.01; ***, p < 0.001).

Figure 8

The information of donors, histological results and IHC results in human heart tissues. (A) Age of donors. (n = 6-8) (B) Abdominal fat thickness of donors. (n = 6-8) (C) Heart weight of donors. (n = 6-8) (D) HE staining and cardiomyocyte size. (n = 6-8; Scale bar = 400 μm) (E) Sirus red staining and collagen area ratio. (n = 3; Scale bar = 800 μm) (F) PAS staining and positive area ratio. (n = 3; Scale bar = 400 μm) (G) IHC images and relative quantification of FABP3. (n = 4-5; Scale bar = 200 μm) (H) IHC images and relative quantification of CPT-1α. (n = 4-7; Scale bar = 200 μm) (I) IHC images and relative quantification of ATGL. (n = 3; Scale bar = 200 μm) (J) IHC images and relative quantification of p-GSK-3β. (n = 3-4; Scale bar = 200 μm) (K) IHC images and relative quantification of HO-1. (n = 5-8; Scale bar = 200 μm) (L) IHC images and relative quantification of NQO1. (n = 3-4; Scale bar = 200 μm) Data are expressed as mean ± SD. (*, p<0.05; **, p<0.01; ***, p<0.001; ##, p<0.01; ###, p<0.001.)