The Effect And Mechanism Of Finerenone In Diabetic Cardiomyopathy

BackgroundDiabetes is a growing global disease,with more than 500 million people suffering from it according to statistics.Among these patients,without vascular disease and other cardiac abnormalities,heart failure develops,called diabetic cardiomyopathy(DCM).The main features of DCM are abnormal myocardial lipid metabolism,apoptosis,fibrosis and myocardial remodeling.However,patients with diabetes and obesity have significantly higher levels of myocardial fatty degeneration and are more prone to impaired cardiac function.Excess free fatty acids(FFA)in the blood may lead to increased cardiac FFA uptake and increased intracellular fatty acid(FA),leading to lipotoxicity of downstream metabolites such as reactive oxygen species(ROS).Excessive activation of the renin-angiotensin-aldosteronesystem(RAAS)can be seen in DCM,aldosterone levels in diabetic patients are higher than normal people,the activation of aldosterone can cause cardiomyocyte fibrosis,and aldosterone can also cause apoptosis through the mitochondrial pathway.Finerenone is the third generation of mineralocorticoid receptor antagonists(MRAs).Compared with the previous MRAs,finerenone has great specificity for mineralocorticoid receptor(MR),which gives finerenone great application potential.In large-scale clinical trials,finerenone has demonstrated strong cardiac benefits,including reduced cardiovascular mortality and reduced cardiac fibrosis.However,the effect of finerenone on DCM and its related molecular mechanism,especially in steatosis and apoptosis,have not been reported yet.In this study,we analyzed the changes in the transcriptome level of cardiomyocytes under finerenone treatment,and studied the possible mechanism of finerenone’s protective effect through further cell and animal experiments.Objective(1)A stable rat model of type Ⅱ.diabetes mellitus was established,and finerenone was given to explore the effect and mechanism of finerenone treatment on cardiac function in diabetic rats.(2)Neonatal rat cardiomyocytes(NRCMs)were extracted for cell culture,and explore the effect and molecular mechanism of finerenone on cardiomyocyte metabolism and apoptosis stimulated by high glucose and high fatty acid.Method1.Animal modelEighteen 8-week-old Sprague-Dawley rats were randomly divided into 3 groups(n=6).All rats were maintained on a light-dark cycle at 23℃.The control group was fed a basic diet(12.79%fat,20.54%protein,66.67%carbohydrate),and the rest were fed a high-fat diet(47.18%fat,19.54%protein,33.29%carbohydrate).Then intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)were performed to identify insulin-resistant rats.A single intraperitoneal injection of streptozotocin(40 mg/kg)was performed on insulinresistant rats after a high-fat diet to induce a type 2 diabetes model.Fasting blood glucose(FBG)was measured 7 days after injection,and rats with FBG≥11.1 mmol/L were considered as successful diabetes models.After 8 weeks of hyperglycemia,the drug group was treated with finerenone(1mg/kg/day).All rats were sacrificed after 8 weeks of finerenone treatment,and all experimental protocols were legally approved by the Animal Care Committee of Shandong University.2.Cardiac functionCardiac function in rats was measured by the Vevo2100 imaging system with an MX250 probe(VisualSonics,Toronto,Canada).The echocardiography parameters involved the left ventricular ejection fraction(LVEF),peak E to peak A ratio(E/A),early(e’)to late(a’)diastolic velocity ratio(e/a)and the fractional shortening(FS).3.Histology stainingHearts were fixed with 4%paraformaldehyde,embedded in paraffin,and cut into 4 μm sections for haematoxylin and eosin(HE)staining.We performed Masson’s trichrome to measure the level of fibrosis.4.Cell treatmentNeonatal rat cardiomyocytes(NRCMs)were extracted for cell culture,and the medium used was dulbecco’s modified eagle medium(DMEM)(5%horse serum,8%calf serum,0.1 mM 5-Bromodeoxyuridinc).Hyperlipidemia or hyperglycemia regulation was mimicked by incubation with high concentrations of common saturated FFA(sodium palmitate,16:0,0.3 mM sigma)or glucose(D-glucose,33 mM).Palmitate was previously conjugated with Bovine Serum Albumin(BSA)in a 3:1 molar ratio as published elsewhere.In control cells,BSA was added as described but in the absence of palmitate.Finerenone(20nM)was added 1 h before stimulation.5.RNA-seq and transcriptome analysisThe total RNA from the NRCMs was extracted according to the instruction manual of the TRIZOI Reagent(Life technologies,California,USA).RNA concentration and purity was measured using NanoDrop 2000(Thermo Fisher Scientific,Wilmington,DE).RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system(Agilent Technologies,CA,USA).After enrichment of mRNA,the cDNA library was constructed,followed by bulk RNA sequencing and transcriptome analysis on the Illumina Novaseq system,entrusted to Biomarker Technologies Inc.(Beijing,China).Differential expression analysis of two groups was performed using the DESeq2.DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution.The resulting P values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate.Genes with an adjusted P-value<0.05&Fold Change≥1.5 found by DESeq2 were assigned as differentially expressed.6.Measurement of intracellular ROSOxidative stress was evaluated via detecting the production of intracellular ROS in cultured NRCMs after different treatments.Cells were incubated with 20 μM 2’,7’dichlorofluorescein-diacetate(DCFH-DA,Solarbio)for 15 minutes and then washed three times with phosphate-buffered saline for removing residual DCFH probe.The fluorescence intensity was observed under an inverted fluorescence microscope(Nikon).7.Examination of apoptosisTUNEL and Annexin V/PI staining were used to measure cell apoptosis.For DNA fragmentation,TUNEL assays kit(Roche,USA)was used to treat the NRCMs or tissue sections according to manufacturer’s instructions.Images were acquired via a fluorescence microscopy(Nikon).FITC Annexin V Apoptosis Detection K it I(BD,USA)was used to determine the stage of cell apoptosis.Data were acquired by flow cytometry(FACSCalibur,BD,US A).8.Immunofluorescence microscopyImmunofluorescence was used to visualize the expression and localization of target proteins.NRCMs were fixed with 4%paraformaldehyde,permeabilized,and then blocked with 2%BSA for 2 hours.Thereafter,cells were incubated with mouse anti-TNFα(Proteintech,60291-1-1g)and rabbit anti-TNFR1(Proteintech,21574-1-AP)antibodies overnight at 4℃.After incubation with two different fluorescent secondary antibodies for 60 minutes at 37℃and staining with DAPI,images were obtained by observation with a confocal fluorescence microscope(LSM710,CarlZeiss,Germany).9.Western blotCardiac tissue or cells were lysed with RIPA lysis buffer.The prepared protein samples were separated by 12%sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDSPAGE)and then transferred to PVDF membrane(Millipore,USA).5%nonfat milk was used for blocking at room temperature for 1 hour,and then the membrane was incubated with 1 antibody at 4℃ overnight.The next day,incubation with secondary antibodies was performed for 1 hour at room temperature before enhanced chemiluminescence using an Amersham Imager 800.10.Statistical analysisAll analyzes were carried out in Prism 6.0(Graphpad)and SPSS 20.0.One-way ANOVA was used to compare the differences between groups and unpaired t-test was used for the differences between two groups.Each experiment was repeated at least three times,and data are shown as mean ± standard deviation(SD).Two-tailed P<0.05 was considered statistically significant.Result1.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.Renal function(urea nitrogen,creatinine,albumin)and liver function(aspartate and alanine aminotransferase)injury markers remained within normal ranges in all groups.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,and the diameter of cardiomyocytes was significantly increased.Finerenone treatment significantly reduced cardiomyocyte diameter in rats compared with the untreated group.The heart function of diabetic rats was examined by ultrasound.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.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.The area of fibrosis among cardiomyocytes increased,and the expressions of collagen I and collagen III increased significantly.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,and the apoptosis marker proteins Cleaved-Caspase3 and Bax/Bcl-2 both increased.After finerenone treatment,the level of myocardial apoptosis decreased.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.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.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.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.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 downregulated.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,posttranslational 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.KEGG pathway enrichment analysis of differential genes showed that the inflammatory signaling pathways TNFα,IL-17,etc.were upregulated in the HF+HG stimulation group compared with the control group,and these pathways were all down-regulated in the treatment group.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.6.Finerenone improves cardiomyocyte metabolism and reduces ROS generation through PPARy/CD36 pathway.Cardiomyocyte apoptosis in DCM may be caused by excessive uptake and accumulation of FFA.The major FFA protein transporter is CD36.Under the stimulation of high glucose and high fatty acid,the expression of PPARγ and CD36 increased,and the expression of PPARy and CD36 decreased after finerenone treatment.At the same time,after the cells take in excess lipids,the formation of ROS will be increased.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.This suggests that finerenone may improve cardiomyocyte metabolism through the PPARy/CD36 pathway.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 TNFa pathway was up-regulated in the high-glucose and high-fat stimulation group,and the TNFa 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.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.This indicates that finerenone relieves cardiomyocyte apoptosis under high glucose and high fat stimulation via the TNFα/TNFR1/CASPASE8 pathway.Conclusion(1)Finerenone treatment did not produce significant burden on the kidneys and liver,and did not significantly reduce blood glucose and lipid levels in diabetic rats.(2)After the treatment of finerenone,cardiac hypertrophy and cardiac dysfunction in diabetic rats were significantly alleviated.(3)Finerenone treatment alleviated myocardial fibrosis and apoptosis in diabetic rat hearts.(4)High-glucose and high-fat stimulation causes a series of inflammatory and metabolic gene changes,which are alleviated by finerenone.(5)Finerenone reduces the level of apoptosis which were induce by HF+HG in NRCMs.(6)Finerenone improves myocardial lipid metabolism abnormalities and reduces ROS production through the PPARy/CD36 pathway.(7)Finerenone reduces cardiomyocyte apoptosis via the TNFα/TNFR1/CASPASE8 pathway.