Dapagliflozin Mediates Plin5/PPARα Signaling Axis to Attenuate Cardiac Hypertrophy

Abstract

Objective: The purpose of this study was to investigate the effect of dapagliflozin (DAPA), a sodium-glucose cotransporter 2 inhibitor, on relieving cardiac hypertrophy and its potential molecular mechanism. Methods: Cardiac hypertrophy induced by abdominal aortic constriction (AAC) in mice, dapagliflozin were administered in the drinking water at a dose of 25 mg/kg/d for 12 weeks was observed. Echocardiography was used to detect the changes of cardiac function, including LVEF, LVFS, LVEDd, LVEDs, HR and LV mass. Histological morphological changes were evaluated by Masson trichrome staining and wheat germ agglutinin (WGA) staining. The enrichment of differential genes and signal pathways after treatment was analyzed by gene microarray cardiomyocyte hypertrophy was induced by AngII (2 μM) and the protective effect of dapagliflozin (1 μM) was observed in vitro. The morphological changes of myocardial cells were detected by cTnI immunofluorescence staining. ELISA and qRT-PCR assays were performed to detect the expressions levels of cardiac hypertrophy related molecules. Results: After 12 weeks of treatment, DAPA significantly ameliorated cardiac function and inhibited cardiac hypertrophy in AAC-induced mice. In vitro, DAPA significantly inhibited abnormal hypertrophy in AngII-induced cardiacmyocytes. Both in vivo and in vitro experiments have confirmed that DAPA could mediate the Plin5/PPARα signaling axis to play a protective role in inhibiting cardiac hypertrophy. Conclusion: Dapagliflozin activated the Plin5/PPARα signaling axis and exerts a protective effect against cardiac hypertrophy.

Keywords: AngII; Plin5/PPARα signaling; cardiac hypertrophy; dapagliflozin; mice.

FIGURE 1

Dapagliflozin alleviated cardiac remodeling and improved cardiac function in AAC-induced mice (A) Representative M-mode echocardiography images. In AAC-induced mice, dapagliflozin improved ejection fractions (EF) (B) and fractional shortening (FS) (C), LV mass (D), Heart rate (E), LV end-diastolic volume, LVEDV (F), LV end-systolic volume, LVESV, n = 8 (G). Images of Masson-stained (H) and WGA-stained sections (I) in different groups, n = 3, original magnification, 400× (J) Images of whole heart from mice in different groups after 28 days. (K) The heart weight to body weight ratio was determined, n = 8. The data were expressed as mean ± SD, *p < 0.05, vs. sham group; #p < 0.05, vs. AAC group.

FIGURE 2

Effect of dapagliflozin on the level of cardiac hypertrophy-related genes in AAC-induced mice. The contents of ANP (A), BNP (B), β-MHC (C) and cTnI (D) in cardiac tissues of different groups were detected via ELISA kits. qRT-PCR analysis was performed to detect the mRNA expression of ANP (E), BNP (F) and β-MHC (G). The data were expressed as mean ± SD, *p < 0.05, vs. sham group; #p < 0.05, vs. AAC group.

FIGURE 3

Dapagliflozin attenuated AngII-induced cardiomyocyte hypertrophy. (A) The morphological changes of cardiomyocyte cells were evaluated by immunofluorescence staining with anti-cTNI (red), cells were counterstained with DAPI (blue) staining the nucleus, Phalloidin-FITC staining the cytoskeleton (green) qRT-PCR analysis was used to detect the mRNA expression of Nppa (B), Nppb (C), Serca2 (D) and myh7 (E) in different groups. The data were expressed as mean ± SD in three independent experiments, n = 3, *p < 0.05, **p < 0.01, vs. control group; #p < 0.05, ##p < 0.01, vs. AngII group.

FIGURE 4

Differential gene analysis of dapagliflozin in AAC-induced mice detected by gene microarray (A) Clustered heat map showed the differential gene expression profile after dapagliflozin treatment in AAC-induced mice. (B) Signaling pathway enrichment analysis among different groups, the PPAR signal pathway (red line) is the signal pathway of this research.

FIGURE 5

Dapagliflozin mediated the Plin5/PPARα signaling axis to mitigate AngII-induced cardiomyocyte hypertrophy. qRT-PCR analysis was used to detect the mRNA expressions of STAT1 (A), Plin5 (B), PPARα (C), HMGCS2 (D) and PDK4 (E) in cardiomyocytes of different groups. (F–J) Western blotting assay was performed to detect the protein expressions of STAT1, Plin5, PPARα, HMGCS2 and PDK4. The data were expressed as mean ± SD, n = 3, *p < 0.05, vs. control group; #p < 0.05, vs. AngII group.

FIGURE 6

Dapagliflozin mediated the Plin5/PPARα signaling axis to attenuate cardiac hypertrophy in vivo. qRT-PCR analysis was used to detect the mRNA expressions of STAT1 (A), Plin5 (B), PPARα (C), HMGCS2 (D) and PDK4 (E) in cardiac tissues. (E) Western blotting assay was performed to detect the protein expressions of STAT1, Plin5, PPARα, HMGCS2 and PDK4. The data were expressed as mean ± SD, n = 3, *p < 0.05, vs. sham group; #p < 0.05, vs. AAC group.

FIGURE 7

Silence Plin5 or GW6471 could reverse the protective effect of dapagliflozin-mediated PPARα signaling axis in AngII-induced cardiomyocyte hypertrophy. The content of ANP (A), BNP (B), β-MHC (C) and cTNI (D) in cell culture supernatant was detected by ELISA kits. qRT-PCR analysis was performed to detect the mRNA expression of Nppa (E), Nppb (F), Serca2 (G) and myh7 (H) in different groups. (I–L) Western blotting assay was used to detect the protein expressions of Plin5, PPARα, HMGCS2 and PDK4 in cardiomyocyte of different groups. The data were expressed as mean ± SD, n = 3, *p < 0.05, vs. AngII + DAPA + DMSO group; #p < 0.05, vs. AngII + DAPA + si-NC group.