SGLT2 inhibitor downregulates ANGPTL4 to mitigate pathological aging of cardiomyocytes induced by type 2 diabetes

Abstract

Background: Senescence is recognized as a principal risk factor for cardiovascular diseases, with a significant association between the senescence of cardiomyocytes and inferior cardiac function. Furthermore, type 2 diabetes exacerbates this aging process. Sodium-glucose co-transporter 2 inhibitor (SGLT2i) has well-established cardiovascular benefits and, in recent years, has been posited to possess anti-aging properties. However, there are no reported data on their improvement of cardiomyocytes function through the alleviation of aging. Consequently, our study aims to investigate the mechanism by which SGLT2i exerts anti-aging and protective effects at the cardiac level through its action on the FOXO1-ANGPTL4 pathway.

Methods: To elucidate the underlying functions and mechanisms, we established both in vivo and in vitro disease models, utilizing mice with diabetic cardiomyopathy (DCM) induced by type 2 diabetes mellitus (T2DM) through high-fat diet combined with streptozotocin (STZ) administration, and AC16 human cardiomyocyte cell subjected to stimulation with high glucose (HG) and palmitic acid (PA). These models were employed to assess the changes in the senescence phenotype of cardiomyocytes and cardiac function following treatment with SGLT2i. Concurrently, we identified ANGPTL4, a key factor contributing to senescence in DCM, using RNA sequencing (RNA-seq) technology and bioinformatics methods. We further clarified ANGPTL4 role in promoting pathological aging of cardiomyocytes induced by hyperglycemia and hyperlipidemia through knockdown and overexpression of the factor, as well as analyzed the impact of SGLT2i intervention on ANGPTL4 expression. Additionally, we utilized chromatin immunoprecipitation followed by quantitative real-time PCR (ChIP-qPCR) to confirm that FOXO1 is essential for the transcriptional activation of ANGPTL4.

Results: The therapeutic intervention with SGLT2i alleviated the senescence phenotype in cardiomyocytes of the DCM mouse model constructed by high-fat feeding combined with STZ, as well as in the AC16 model stimulated by HG and PA, while also improving cardiac function in DCM mice. We observed that the knockdown of ANGPTL4, a key senescence-promoting factor in DCM identified through RNA-seq technology and bioinformatics, mitigated the senescence of cardiomyocytes, whereas overexpression of ANGPTL4 exacerbated it. Moreover, SGLT2i improved the senescence phenotype by suppressing the overexpression of ANGPTL4. In fact, we discovered that SGLT2i exert their effects by regulating the upstream transcription factor FOXO1 of ANGPTL4. Under conditions of hyperglycemia and hyperlipidemia, compared to the control group without FOXO1, the overexpression of FOXO1 in conjunction with SGLT2i intervention significantly reduced both ANGPTL4 mRNA and protein levels. This suggests that the FOXO1-ANGPTL4 axis may be a potential target for the cardioprotective effects of SGLT2i.

Conclusions: Collectively, our study demonstrates that SGLT2i ameliorate the pathological aging of cardiomyocytes induced by a high glucose and high fat metabolic milieu by regulating the interaction between FOXO1 and ANGPTL4, thereby suppressing the transcriptional synthesis of the latter, and consequently restoring cardiac function.

Keywords: ANGPTL4; Cellular senescence; Diabetic cardiomyopathy; FOXO1; SGLT2i.

Fig. 1

SGLT2 Inhibitors Ameliorate Cardiac Dysfunction and Cellular Senescence in DCM Mice Induced by High-Fat Diet and STZ. A Treatment with SGLT2 inhibitors in DCM mice model induced by a high-fat diet and STZ. B Representative echocardiographic images of mouse hearts. C and D Levels of left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS). E Representative H&E staining (top) and Masson’s trichrome staining (bottom) of cardiac sections; scale bar, 20 mm. F and G Expression levels of IL-6 and IL-1β in serum detected by ELISA. H–J Detection of P53 and P21 by Western blotting. K and L Immunofluorescent staining of γ-H2AX (green) in mouse hearts; scale bar, 20 mm. Data are presented as mean ± SEM; *p < 0.05 **p < 0.01, ***p < 0.001

Fig. 2

SGLT2 Inhibitors Mitigate Cellular Senescence in Human Ventricular Cardiomyocyte cell (AC16) Exposed to High Glucose(HG)and Palmitic Acid(PA). A–D AC16 cells were treated with LG(1mmol/L) and HG(33.3mmol/L) + PA(100µM) for 48 h, followed by incubation with SGLT2 inhibitors (0.5µM) for an additional 48 h. P53 and P21 proteins in cell lysates were detected by Western blotting. E and F β-galactosidase staining(β-gal) of AC16 cells; scale bar, 20 mm. G and H Immunofluorescent staining of γ-H2AX (green) in AC16 cells; scale bar, 20 mm. Data are presented as mean ± SEM; *p < 0.05 **p < 0.01, ***p < 0.001

Fig. 3

Different Expression Genes (DEGs) in Cardiac Muscle Tissue from DCM Mice and WT Mice were Tested by RNA-Sequence Analysis (n = 3). A and B Volcano plots and heatmaps display the normalized gene expression values from RNA-seq of myocardium in DCM and WT mice. C Functional enrichment analysis of DEGs in myocardium of DCM and WT mice. D Venn diagrams of overlapping genes between RNA-sequence analysis data and the CellAge database, including 20genes: ANGPTL4,CENPQ, LSM3,IMPA2,FKBP5,PSRC1,UBE2T, DIO2,CXCL10,MX1,IFIT3,E2F8,MYBL2,IFIT2,CDCA7,IS–G15,SYT1,IFIT1,CCNG1,EEF1E1

Fig. 4

Enrichment analyses using gene set enrichment analysis (GSEA)of RNA-sequence analysis. A DNA damage/telomere stress induced senescence. B Senescence-associated secretory phenotype (SASP). C Assembly of the oRC complex at the origin of replication. D Diseases of programmed cell death. E DNA methylation. F DNA Replication Pre-Initiation. G DNA Replication. H Inhibition of DNA recombination at telomere. I Mitochondrial translation

Fig. 5

Knockdown of ANGPTL4 alleviates HG +PA-Induced cellular senescence in cardiomyocytes. (A-D) P53 and P21 proteins in cell lysates were detected by Western blotting. (E and F) β-gal staining of treated AC16 cells; scale bar, 20 mm. (G and H) Immunofluorescent staining of γ-H2AX (green) in treated AC16 cells; scale bar, 20 mm. Data are presented as mean ± SEM; *p < 0.05 **p < 0.01, ***p < 0.001

Fig. 6

Overexpression of ANGPTL4 Exacerbates HG + PA-Induced Cellular senescence in cardiomyocytes. (A-D) P53 and P21 proteins in cell lysates were detected by Western blotting. (E and F) β-gal staining of treated AC16 cells; scale bar, 20 mm. (G and H) Immunofluorescent staining of γ-H2AX (green) in treated AC16 cells; scale bar, 20 mm. Data are presented as mean ± SEM; *p < 0.05 **p < 0.01, ***p < 0.001

Fig. 7

SGLT2 inhibitors mitigate cellular senescence through ANGPTL4 modulation. A–D P53 and P21 proteins in cell lysates were detected by Western blotting. E and F β-gal staining of treated AC16 cells; scale bar, 20 mm. G and H Immunofluorescent staining of γ-H2AX (green) in treated AC16 cells; scale bar, 20 mm. Data are presented as mean ± SEM; *p < 0.05 **p < 0.01, ***p < 0.001

Fig. 8

SGLT2 Inhibitors Can Suppress the Transcription of ANGPTL4 by Reducing Cardiac FOXO1. A Expression of FOXO1 binding sites in the JASPAR database. B–D AC16 cells were transfected with FOXO1 siRNA (0.5µM) and incubated for 24 and 48 h, respectively, to detect the levels of ANGPTL4 mRNA and its protein. E–G AC16 cells were transfected with an overexpression plasmid of FOXO1 (2 µg/µl) and incubated for 24 and 48 h, respectively, to detect the levels of ANGPTL4 mRNA and its protein. H–J AC16 cells were treated with HG (33.3mmol/L) + PA (100µM) for 48 h, then transfected with an overexpression plasmid of FOXO1 (2 µg/µl) using lipo2000, followed by incubation with SGLT2 inhibitors (0.5µM) for an additional 48 h to detect the levels of ANGPTL4 mRNA and its protein. K Chromatin immunoprecipitation assay using AC16 cells to verify the binding sites of FOXO1 in the promoter region of ANGPTL4. L Chromatin immunoprecipitation assay using primary mouse cardiomyocytes to verify the binding sites of FOXO1 in the promoter region of ANGPTL4 Data are presented as mean ± SEM; *p < 0.05 **p < 0.01, ***p < 0.001