Finerenone attenuates myocardial apoptosis, metabolic disturbance and myocardial fibrosis in type 2 diabetes mellitus

Basic characteristic of type 2 diabetic rats

Rats with type 2 diabetes mellitus (T2DM) showed higher levels of blood glucose, free fatty acids, and higher heart weight to body weight ratios. After 8 weeks of finerenone treatment, there was no significant difference in blood glucose and heart weight to body weight ratio, and blood lipids seemed to increase, but this difference did not reach statistical significance (Table 1). Renal function (urea nitrogen, creatinine, albumin) and liver function (aspartate and alanine aminotransferase) injury markers remained within normal ranges in all groups (not shown).

Table 1 Basic information of ratsFinerenone attenuates cardiac hypertrophy and cardiac dysfunction in diabetic rats

Compared with the control group, the hearts of diabetic rats showed the characteristics of pathological hypertrophy (Fig. 1A), and the diameter of cardiomyocytes was significantly increased (Fig. 1B, C). Finerenone treatment significantly reduced cardiomyocyte diameter in rats compared with the untreated group.

Fig. 1figure 1

Finerenone attenuates left ventricular remodeling and improves cardiac function in diabetic rats. A General and heart morphology photos of rats. B HE staining of heart. B1 HE staining of cross shaft of musculi papillares in heart. B2 Representative HE staining of longitudinal left ventricular (LV) sections. B3 Representative HE staining of LV transverse sections. C Quantitative analysis of the cardiomyocyte diameter in heart tissue. D Representative images of 2D echocardiograms (D1), M‐mode echocardiograms (D2), Pulse‐wave Doppler echocardiograms of mitral inflow (D3), tissue Doppler echocardiograms (D4). E Assessment of cardiac function, including left ventricular ejection fraction (LVEF), fractional shortening (FS), early to late mitral diastolic blood ratio (E/A), and diastolic mitral annular velocity ratio (E′/A′). *P < 0.05 compared with control; #P < 0.05 compared with DM; Data are means ± SD

The heart function of diabetic rats was examined by ultrasound (Fig. 1D, E). Compared with the control group, the LVEF, F/S, E/A, Eʹ/Aʹ indexes of diabetic rats were abnormal, showing left ventricular systolic and diastolic dysfunction. Cardiac dysfunction was improved in finerenone-treated diabetic rats compared with the untreated group.

Finerenone attenuates myocardial fibrosis and apoptosis in the heart of diabetic rats

Compared with the control group, Masson showed that the level of extracellular matrix in the interstitial area was increased in the diabetic group (Fig. 2A). The area of fibrosis among cardiomyocytes increased, and the expressions of collagen I and collagen III increased significantly (Fig. 2B). Myocardial fibrosis levels were reduced after finerenone treatment. At the same time, TUNEL showed that the apoptosis level of cardiomyocytes in the diabetic group increased (Fig. 2C), and the apoptosis marker proteins Cleaved-Caspase3 and BAX/BCL-2 both increased (Fig. 2D). After finerenone treatment, the level of myocardial apoptosis decreased.

Fig. 2figure 2

Finerenone attenuates myocardial fibrosis and apoptosis in the heart of diabetic rats. A Masson’s trichrome staining of myocardial mesenchyme and peripheral vessel. B Representative images of the Western blot of collagen I and collagen III, with the corresponding analysis. C Cell apoptosis as determined by TUNEL assay with the corresponding analysis: apoptosis cell stained red; nuclei stained blue with DAPI. D Representative images of the Western blot of Cleaved-Casp3, BAX and BCL-2 with the corresponding analysis. *P < 0.05 compared with control; #P < 0.05 compared with DM; Data are means ± SD

Finerenone reduces the apoptosis of cardiomyocytes stimulated by high glucose and high fatty acid

After the primary cardiomyocytes were stimulated by high fatty acid (HF) and high glucose (HG), the level of apoptosis was significantly increased, and the ratio of BAX/BCL-2, Cleaved-Caspase3/GAPDH increased. The ratio of BAX/BCL-2 and Cleaved-Caspase3/GAPDH was significantly decreased after finerenone treatment in NRCMs (Fig. 3C). TUNEL test showed that under the stimulation of HF and HG, the proportion of TUNEL positive cells increased, and the proportion of positive cells decreased after finerenone treatment (Fig. 3B). At the same time, we also performed Annexin V/PI staining and detected by flow cytometry. Compared with the control group, after HF and HG stimulation, the proportion of cells in the early stage of apoptosis (Annexin V+) and late stage of apoptosis (Annexin V+/PI+) increased, and the overall percentage of apoptotic cells increased (Fig. 3A). After treatment of finerenone, the proportion of early apoptosis and late apoptosis decreased, suggesting that finerenone has a protective effect on apoptosis induced by HF and HG stimulation.

Fig. 3figure 3

Finerenone attenuates apoptosis in neonatal rat myocardium. A Flow cytometry and quantification of apoptotic NRCMs with the corresponding analysis. B NRCMs apoptosis as determined by TUNEL assay with the corresponding analysis: apoptosis cell stained red; nuclei stained blue with DAPI. C Representative images of the Western blot of Cleaved-Casp3, BAX and BCL-2 in NRCMs with the corresponding analysis. *P < 0.05 compared with NC; #P < 0.05 compared with HF + HG; Data are means ± SD

Transcriptome data suggest finerenone improves cardiomyocytes in multiple ways

By RNA-seq and transcriptome analysis, 734 genes were altered between control (Group NC) and HF + HG stimulated (Group S) cells under differential conditions (set fold change > 1.5, P-value < 0.05). Compared with the HF + HG group treated with finerenone (Group SD), a total of 1069 genes were changed, 184 genes were up-regulated, and 885 genes were down-regulated (Fig. 4A–C). The COG database was used to classify and count the differential genes. Compared with the control group, after HF + HG stimulation, the signal transduction mechanism, post-translational modification, lipid transport and metabolism and other genes were changed. Compared with the experimental group, the changed genes were concentrated in signal transduction mechanism, post-translational modification, amino acid transport and metabolism, lipid transport and metabolism in the treatment group (Fig. 4D). KEGG pathway enrichment analysis of differential genes showed that the inflammatory signaling pathways TNFα, IL-17, etc. were up-regulated in the HF + HG stimulation group compared with the control group, and these pathways were all down-regulated in the treatment group (Fig. 4E). Taken together, these data suggest that high glucose and high fatty acid stimulation can cause changes in a series of genes related to inflammation and metabolism, and finerenone alleviates these changes.

Fig. 4figure 4

Transcriptome results reveal that finerenone ameliorates high glucose and high fatty acid-induced inflammation and metabolic abnormalities in neonatal rat cardiomyocytes. A The volcano plots. B The MA plot. C The Venn diagram. D COG Function Classification of Consensus Sequence. E KEGG pathway enrichment analysis. Gene function was annotated based on the following databases: Nr (NCBI non-redundant protein sequences); Pfam (Protein family); KOG/COG (Clusters of Orthologous Groups of proteins); Swiss-Prot (A manually annotated and reviewed protein sequence database); KO (KEGG Ortholog database); GO (Gene Ontology). NC (negative control group), S (HF + HG stimulation group), ND (negative control with drug group), SD (HF + HG stimulation with drug group)

Finerenone improves cardiomyocyte metabolism and reduces ROS generation through PPARγ/CD36 pathway

Cardiomyocyte apoptosis in DCM may be caused by excessive uptake and accumulation of FFA. The major FFA protein transporter is CD36 [18]. Under the stimulation of high glucose and high fatty acid, the expression of PPARγ and CD36 increased, and the expression of PPARγ and CD36 decreased after finerenone treatment (Fig. 5A). At the same time, after the cells take in excess lipids, the formation of ROS will be increased [19]. Compared with the control group, the ROS in cardiomyocytes was significantly increased under high glucose and high fatty acid stimulation, and the ROS decreased significantly in the finerenone treatment group (Fig. 5B). This suggests that finerenone may improve cardiomyocyte metabolism through the PPARγ/CD36 pathway.

Fig. 5figure 5

Finerenone reduces PPARγ and CD36 expression and reduces ROS generation under high glucose and high fatty acid stimulation in neonatal rat cardiomyocytes. A Representative images of the Western blot of Cleaved-Casp3, BAX and BCL-2 in NRCMs with the corresponding analysis. B The production of ROS with the corresponding analysis. *P < 0.05 compared with NC; #P < 0.05 compared with HF + HG; Data are means ± SD

Finerenone reduces apoptosis in neonatal rat cardiomyocytes stimulated by high glucose and high fatty acid via TNFα/TNFR1 pathway

The transcriptome suggested that, compared with the control group, the TNFα pathway was up-regulated in the high-glucose and high-fat stimulation group, and the TNFα signaling pathway was down-regulated after finerenone treatment. We localized TNFα and TNFR1 by confocal microscope, and found that the expression of TNFα and TNFR1 in cardiomyocytes increased under high glucose and high fatty acid stimulation, and the expression of both decreased after finerenone treatment (Fig. 6A). By western blot, compared with the control group, the expression of TNFα, TNFR1, and Cleaved-Caspase8 in the high glucose and high fatty acid group was up-regulated, and the up-regulation of these proteins was alleviated after treatment with finerenone (Fig. 6B). This indicates that finerenone relieves cardiomyocyte apoptosis under high glucose and high fat stimulation via the TNFα/TNFR1/CASPASE8 pathway.

Fig. 6figure 6

Finerenone reduces apoptosis in neonatal rat cardiomyocytes stimulated by high glucose and high fatty acid via TNFα/TNFR1 pathway. A Confocal images of TNFA and TNFR1 with the corresponding analysis. B Representative images of the Western blot of TNFα, TNFR1 and Cleaved-Caspase8 in NRCMs with the corresponding analysis. *P < 0.05 compared with NC; #P < 0.05 compared with HF + HG; Data are means ± SD

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