Multi-omics insights into potential mechanism of SGLT2 inhibitors cardiovascular benefit in diabetic cardiomyopathy

Abstract

Background: Metabolic and energy disorders are considered central to the etiology of diabetic cardiomyopathy (DCM). Sodium-glucose cotransporter-2 inhibitors (SGLT2i) can effectively reduce the risk of cardiovascular death and heart failure in patients with DCM. However, the underlying mechanism has not been elucidated.

Methods: We established a DCM rat model followed by treatment with empagliflozin (EMPA) for 12 weeks. Echocardiography, blood tests, histopathology, and transmission electron microscopy (TEM) were used to evaluate the phenotypic characteristics of the rats. The proteomics and metabolomics of the myocardium in the rat model were performed to identify the potential targets and signaling pathways associated with the cardiovascular benefit of SGLT2i.

Results: The diabetic rat showed pronounced DCM characterized by mitochondrial pleomorphic, impaired lipid metabolism, myocardial fibrosis, and associated diastolic and systolic functional impairments in the heart. To some extent, these changes were ameliorated after treatment with EMPA. A total of 43 proteins and 34 metabolites were identified as targets in the myocardium of diabetic rats treated with EMPA. The KEGG analysis showed that arachidonic acid is associated with the maximum number of related pathways and may be a potential target of EMPA treatment. Fatty acid (FA) metabolism was enhanced in diabetic hearts, and the perturbation of biosynthesis of unsaturated FAs and arachidonic acid metabolism was a potential enabler for the cardiovascular benefit of EMPA.

Conclusion: SGLT2i ameliorated lipid accumulation and mitochondrial damage in the myocardium of diabetic rats. The metabolomic and proteomic data revealed the potential targets and signaling pathways associated with the cardiovascular benefit of SGLT2i, which provides a valuable resource for the mechanism of SGLT2i.

Keywords: SGLT2 inhibitors; diabetic cardiomyopathy; mechanisms; metabolomics; proteomics.

FIGURE 1

The study design and phenotypic characteristics of the rats after STZ intraperitoneal injection. (A) Schematic of the study design. (B,C) The body weight (B) and blood glucose (C) of rats in the control, high glucose (HG), and empagliflozin (EMPA) group. (a) comparison is significant for Control vs. HG, (b) comparisons are significant for HG vs. EMPA, and (c) is significant for Control vs. EMPA. (D) Transmission electron microscopy was observed on the rat heart of the three groups. 5,000x (upper part); 20,000x (lower part). (E) Representative photomicrograph of H and E staining (upper part) and Masson’s staining (lower part) of the myocardium, 200x (insert 400x).

FIGURE 2

In three groups, echocardiographic measurements of the rats at 18 weeks and 30 weeks after STZ injection. (A) Representative M-mode tracings of rats in three groups (n = 6 for the control group, n = 10 for the high glucose (HG) group, and n = 8 for the empagliflozin (EMPA) group). (B) Plots of echocardiographic measurement results for left ventricle internal dimension at end-diastole/systole (LVID;d/s), left ventricle posterior wall thickness at end-diastole (LVPW’d), and ejection fraction (EF) percentage, upper part for 18 weeks, the lower part for 30 weeks. *p < 0.05, ^**p < 0.01.

FIGURE 3

The protein expression changes in the myocardium. (A) Volcano plots of protein expression changes. Cut-off value: p < 0.05, fold change (FC) ≥ 1.5 (upregulation) or ≤ 0.67 (downregulation), the red and green point represents the upregulated and downregulated proteins, respectively. (B) C-means cluster diagram of differential proteins. The proteins are classified into 6 clusters according to their expression levels in each group, and the horizontal coordinate is the group, the vertical coordinate is the protein expression level (correction Z-value), each line represents one protein, larger membership values indicate that the protein is closer to the average level protein of this cluster. (C) Hierarchical clustering analysis of differential in empagliflozin (EMPA) and high glucose (HG) group. (D) GO enrichment of differential proteins in EMPA and HG groups. BP, biological process; CC, cellular component; and MF, molecular function.

FIGURE 4

The differential metabolite of the myocardium. (A,B) Volcano plots of differential metabolite. (A) High glucose (HG) group vs. control group; (B) empagliflozin (EMPA) group vs. HG group. Cut-off value: p < 0.05, fold change (FC) > 1.2 (upregulation) or < 0.67 (downregulation), the red and green point represents the upregulated and downregulated proteins, respectively. (C) Hierarchical clustering analysis of differential metabolite of myocardium in three groups. (D) The correlation diagram of differential metabolites in groups EMPA and HG (top 20). The blue point represents a negative correlation (negative value), and the red represents a positive correlation (positive value). (E) Receiver operating characteristic (ROC) curves of top 5 metabolites [top 5 areas under the curve (AUC)]. The vertical coordinate is the true positive rate (sensitivity), and the horizontal coordinate is the false positive rate (1—specificity).

FIGURE 5

Combined analysis of differential proteins and differential metabolites. (A,B) KEGG enrichment bubble diagram of differential proteins (A) and differential metabolites (B) in groups EMPA and HG. (C) Heat map of correlation between differential protein and differential metabolite expression. The horizontal axis represents proteins, and the vertical axis represents metabolites. (D) Combined analysis of pathway enrichment of differential protein and metabolites. The yellow square represents the pathways shared by differential metabolites and differential proteins, and the circles and triangle represent the differential proteins and differential metabolites involved in the pathway, respectively. Circles represent differential proteins, triangles represent differential metabolites, red is represent up-regulated and green is down-regulated.