4.1 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).
Table1. Basic information of rats. TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein Cholesterol; TG, total triglyceride; FFA, free fatty acids; GLU, glucose; ALD, aldosterone; HW, heart weight.
|
Control
|
DM
|
DM+Finerenone
|
TC (mmol/L)
|
1.78±0.28
|
2.38±0.32*
|
3.31±1.51
|
HDL-C (mmol/L)
|
1.18±0.21
|
0.98±0.22
|
1.01±0.39
|
LDL-C (mmol/L)
|
0.22±0.06
|
0.75±0.38*
|
1.24±0.91
|
TG (mmol/L)
|
0.92±0.31
|
3.29±1.22*
|
4.57±3.31
|
FFA (umol/dL)
|
25.88±6.15
|
50.72±12.16*
|
70.55±25.88
|
GLU (mmol/L)
|
8.6±1.67
|
23.47±2.17*
|
25.07±0.85
|
ALD (pg/mL)
|
246.45±86.22
|
219.34±47.17
|
262.95±18.8
|
Weight(g)
|
651.33±91.11
|
465.17±54.84
|
483.83±30.87
|
HW(g)
|
2.24±0.27
|
2.08±0.11
|
2.25±0.3
|
HW/Weight(mg/g)
|
3.45±0.23
|
4.5±0.32*
|
4.63±0.44
|
* P < 0.05 compared with the control group.
4.2 Finerenone 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.
The heart function of diabetic rats was examined by ultrasound (Fig.1D,E). Compared with the control group, the LVEF, F/S, E/A, E'/A' 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.
4.3 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.
4.4 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.
4.5 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.
4.6 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 [17]. 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 [18]. 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.
4.7 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.