Abstract Ketone bodies are considered as an alternative energy source for diabetic cardiomyopathy (DCM) and can improve the energy supply of the heart muscle, suggesting that it may be an important area of research and development as a therapeutic target for DCM. Cumulative cardiovascular trials have shown that sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular events in diabetic populations. Whether SGLT2 inhibitors improve DCM by enhancing ketone body metabolism remains and whether they help prevent oxidative damage remains to be clarified. Here, we present the combined results of nine GSE datasets for diabetic cardiomyopathy ([31]GSE215979, [32]GSE161931, [33]GSE145294, [34]GSE161052, [35]GSE173384, [36]GSE123975, [37]GSE161827, [38]GSE210612, and [39]GSE5606). We found significant up-regulated gene 3-hydroxymethylglutaryl CoA synthetase 2 (HMGCS2) and down-regulated gene 3-hydroxybutyrate dehydrogenase (BDH1) and 3-oxoacid CoA-transferase1 (OXCT1), respectively. Based on the analysis of the constructed protein interaction network, it was found that HMGCS2 was in the core position of the interaction network. In addition, Gene ontology (GO) enrichment analysis mainly focused on redox process, acyl-CoA metabolic process, catalytic activity, redox enzyme activity and mitochondria. The activity of HMGCS2 in DCM heart was increased, while the expression of ketolysis enzymes BDH1 and OXCT1 was inhibited. In vivo, Empagliflozin (Emp) treated DCM group significantly decreased ventricular weight, myocardial cell cross-sectional area, and myocardial fibrosis. In addition, Emp further promoted the activity of BDH1 and OXCT1, increased the utilization of ketone bodies, further promoted the activity of HMGCS2 in DCM, and increased the synthesis of ketone bodies, prevented mitochondrial breakage and dysfunction, increased myocardial ATP to provide sufficient energy, inhibited oxidative stress and apoptosis of cardiac cells ex vivo, and improved the myocardial dysfunction of DCM. Emp can improve mitochondrial dysfunction in diabetic cardiomyopathy by regulating ketone body metabolism and oxidative stress. These findings provide a theoretical basis for evaluating Emp as a treatment for DCM. Keywords: Diabetic cardiomyopathy, SGLT2 inhibitor, Ketone body metabolism, Oxidative stress, Mitochondrial dysfunction Graphical abstract Image 1 [40]Open in a new tab Highlights * • DEGs involved in DCM were obtained and analysed from nine GSE datasets. * • Expression levels of the BDH1 and OXCT1 were elevated in myocardial tissues of the diabetic db/db+Emp group, as was the expression level of HMGCS2. * • Emp improves DCM mitochondrial dysfunction by promoting the expression of key enzymes in ketone body metabolism and increases myocardial ATP to supplement energy. * • Emp alleviates oxidative stress and apoptosis of DCM cardiomyocytes. Abbreviations 3-NT 3-nitrotyrosine 4-HNE 4-hydroxy-2-nonenal AcAc Acetoacetate ACOT1 Acyl-CoA thioesterase 1 ANOVA One-way analysis of variance ANP Atrial natriuretic peptide BDH1 3-hydroxybutyrate dehydrogenase BKs C57BLKs β-OHB β-hydroxybutyrate BP Biological process Ctrl/CTR/CON Control CAT Catalase CC Cellular component CD Control diet CK Control check DAB Diaminobenzidine DAPA-HF Dapagliflozin and Prevention of Advers e Outcomes in Heart Failure DAPI 4′, 6-diamidino-2-phenylindole DAVID Database for Annotation, Visualization and Integrated Discovery DCM Diabetic cardiomyopathy DEGs Differential expression genes DM Ddiabetes mellitus DRP1 Dynamin-associated protein 1 ECL Enhanced chemiluminescence ELISA Enzyme-linked immunosorbent assay Emp Empagliflozin ETC Electron transport chain EMPEROR Empagliflozin Outcome Trial in Patient Reduced s with Chronic Heart Failure and a Reduced Ejection Fraction GEO Gene Expression Omnibus GO Gene ontology GPL GEO Platform GSE GEO series GSH-Px Glutathione peroxidase H&E Hematoxylin-eosin staining HFHS High fat high sucrose HG High glucose HMGCS2 3-hydroxymethylglutaryl CoA synthetase 2 HW/TL Heart weight to tibia length ratio ID Identification IHC Immunohistochemistry IPGTT Intraperitoneal glucose tolerance test ITT Insulin tolerance test KEGG Kyoto Encyclopedia of Genes and Genomes LVEF Left ventricular ejection fraction LVFS Left ventricular fractional shortening MCODE Molecular complex detection MDA Malondialdehyde MF Molecular Function MFN1 Mitofusin 1 mtDNA Mitochondrial DNA NG Normal glucose nDNA Nuclear DNA OPA1 Optic atrophy 1 OXCT1 3-oxoacid CoA-transferase1 PBS Phosphate buffered saline PMID PubMed identification PPAR Peroxisome proliferator-activated receptor PPI Protein-protein interaction ROS Reactive oxygen species SD Standard deviation SGLT2 Sodium-glucose cotransporter 2 SOD Superoxide dismutase STRING Search tool for the retrieval of interacting genes/proteins STZ Streptozotocin T2D Type 2 diabetes TUNEL Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling VEH Vehicle WGA Wheat germ thrombin 1. Introduction Diabetic cardiomyopathy (DCM) is a specific heart disease caused by diabetes, independent of other cardiovascular diseases [[41]1]. The heart is an organ with high energy requirements and metabolic flexibility, so it can produce a variety of energy substrates to produce ATP under different physiological conditions. Impaired metabolic balance and oxidative stress damage have been shown to be an important aspect of DCM. Therefore, maintaining cardiometabolic homeostasis is a promising strategy for the treatment of DCM. Ketone bodies have been shown to be an alternative energy source for heart failure, which can improve myocardial energy supply and thus adapt to heart failure. The traditional view is that ketone bodies are only produced in the liver [[42]2]. However, in recent years, it has also been found that ketone bodies are produced in tumor cells [[43]3], heart [[44]4], kidney [[45]5], central nervous system [[46]6,[47]7], islet beta cells [[48]8] and retinal pigment epithelial cells [[49]9]. Recent studies have reported changes in the expression level of the ketogenic rate-limiting enzyme 3-hydroxymethylglutaryl CoA synthetase 2 (HMGCS2) in DCM [[50]10,[51]11]. In addition, decreased ketone body oxidation was found in the hearts of db/db mice [[52]12]. Other study has also shown that decreased myocardial 3-hydroxybutyrate dehydrogenase (BDH1) expression level is associated with high-fat diet-induced type 2 diabetes [[53]13]. 3-oxoacid CoA-transferase1 (OXCT1) is a rate-limiting ketolysis enzyme. In OXCT1 knockout mice, left ventricular volume increased, ejection fraction decreased, and mitochondrial ultrastructure was disturbed [[54]14]. Those suggested that decreased ketone body oxidation may be a biological marker and therapeutic target for DCM. However, the specific mechanism of ketone metabolism in DCM remains unclear. DCM is one of the leading causes of death in people with diabetes, and there is still a lack of effective treatments. Sodium-glucose cotransporter 2 (SGLT2) inhibitors entered the public eye as an oral drug for the treatment of T2DM. Multiple cardiovascular outcome trials have demonstrated that SGLT2 inhibitors have a proven cardiovascular benefit. Empagliflozin (Emp) was the first SGLT2 inhibitors drug to complete CVOTs [[55]15]. Recently a series of large clinical trials, including the Dapagliflozin and Prevention of Adverse Outcomes in Heart Failure (DAPA-HF), the Empagliflozin Outcome Trial in Patients with Chronic Heart Failure and a Reduced Ejection Fraction (EMPEROR-Reduced) and the Empagliflozin Outcome Trial in Patients with Chronic Heart Failure with Preserved Ejection Fraction (EMPEROR-Preserved), have shown that SGLT2 inhibitors (SGLT2i) significantly reduce the risk of cardiovascular death in heart failure patients with or without diabetes [[56][16], [57][17], [58][18]]. Moreover, the degree of benefit was independent of left ventricular ejection fraction, which further confirmed that SGLT2 inhibits the cardioprotective effect independent of hypoglycemic mechanism, and pushes its cardiovascular benefit to non-diabetic heart failure population. Several mechanisms of beneficial cardiovascular effects of SGLT2 inhibitors have been hypothesized, such as attenuation of glucose toxicity, improvement of cardiac load conditions, amelioration of oxidative stress and improvement of cardiometabolism by increasing ketone bodies [[59]19,[60]20]. Among others, it has been shown that Emp inhibits oxidative stress and myocardial fibrosis by activating Nrf2/ARE signaling [[61]21]. SGLT2i induces mild ketosis, suggesting a possible mechanism of action for its beneficial effect on heart failure in diabetic patients. Recent studies have shown that ketone bodies increase during SGLT2is treatment, which can reduce mortality and heart failure hospitalization in patients with the heart failure with preserved ejection fraction [[62]22,[63]23]. However, the role of ketone body metabolism and oxidative stress in the absence of heart failure in early DCM remains unclear. In addition, the regulation of Emp on DCM ketone metabolism is still lacking. Therefore, in order to explore these issues, this study intends to explore the effects of Emp on myocardial ketone metabolism and oxidative stress in diabetic db/db mice based on the multiple Gene Expression Omnibus (GEO) database and the entry point of myocardial ketone metabolism of DCM. It provides theoretical basis for early diagnosis and treatment of DCM and prevention of heart failure. 2. Material and methods 2.1. Data preparation We searched and downloaded mRNA expression data of DCM from the GEO ([64]https://www.ncbi.nlm.nih.gov/geo/) using the keywords "diabetic cardiomyopathy" and "diabetic heart". The [65]GSE215979, [66]GSE161931, [67]GSE145294, [68]GSE161052, [69]GSE173384, [70]GSE123975, [71]GSE161827, [72]GSE210612 and [73]GSE5606 were selected and downloaded for DEG analysis. All of the above raw data can be freely downloaded from the GEO database. These datasets met the following criteria: (1) the species was Mus musculus and Ratus norvegicus ([74]GSE5606 only); (2) diabetic heart tissue and control tissue samples; (3) samples were replicated at least two times in the experiment. The corresponding platform annotation files from GEO series (GSE) were shown in [75]Table 1. Table 1. GSE datasets used in this study. Study Set GEO Accession Platform ID Abbreviation (Number of Sample) PMID 1 [76]GSE215979 [77]GPL24247 Illumina NovaSeq 6000 Ctrl Vs DM (6) [78]36706988 2 [79]GSE161931 [80]GPL24247 Illumina NovaSeq 6000 BKs Vs db/db (10) [81]35242109 3 [82]GSE145294 [83]GPL19057 Illumina NextSeq 500 CTR Vs T2D (4) [84]33303689 4 [85]GSE161052 [86]GPL21273 HiSeq X Ten CK Vs DCM (6) [87]35173678 5 [88]GSE173384 [89]GPL24247 Illumina NovaSeq 6000 db/+ Vs db/db (10) [90]34368154 6 [91]GSE161827 [92]GPL19057 Illumina NextSeq 500 CD Vs HFHS (8) [93]34169737 7 [94]GSE123975 [95]GPL10787 Agilent-028005 SurePrint G3 Mouse GE 8 × 60 K Microarray CON_VEH Vs CON_STZ (12) [96]32366681 8 [97]GSE210612 [98]GPL21810 Agilent-074809 SurePrint G3 Mouse GE v2 8 × 60 K Microarray Control Vs Diabetic (6) [99]36078109 9 [100]GSE5606 [101]GPL1355 Affymetrix Rat Genome 230 2.0 Array Normal Vs Diabetic (14) [102]17062650 [103]Open in a new tab 2.2. Identification of differentially expressed genes The “Limma”, “DESeq2” and “RobustRankAggreg” package were used to identify differential expression genes (DEGs) between the DCM and the control group. The cutoff criteria were set as Log 2FC (fold change) > 1 and adjusted P < 0.05. We build the deg heatmap using the "Pheatmap" package and use the "ggplot2″ package to map the DEGs volcano. RobustRankAggreg method was used to integrate and analyze nine datasets [104]GSE215979, [105]GSE161931, [106]GSE145294, [107]GSE161052, [108]GSE173384, [109]GSE123975, [110]GSE161827, [111]GSE210612 and [112]GSE5606, and the common DEGs was obtained. The list of upregulated and downregulated genes in each dataset was sorted by log2FC. The list of all genes was then integrated through the RobustRankAggreg package. 2.3. Animals models All 8-week-old male diabetic db/db (BKs Cg-m +/+ Leprdb/J, a genetic mouse with spontaneous T2DM) and non-diabetic heterozygous db/m mice (n = 10 for each group) with body mass of 20–22 g (Si Pei Fu, China) were fed standard diet freely in SPF-grade animal house at room temperature (22 ± 2) °C, light/dark cycle for 12 h. They were divided into db/m, db/db and db/db + Emp groups, in which the db/db + Emp group was treated with drinking water containing Emp (10 mg/kg/day) for 8 weeks, body weight was measured after 2 months of treatment, and blood samples of each group were collected. Mice in each group were intraperitoneally injected with tribromoethanol (Avitine; 50 mg/kg) were sacrificed, the heart was removed, blood was taken for serum separation, and β-hydroxybutyrate (β-OHB) (Mlbio, China) and acetoacetate (AcAc) (Mlbio, China) levels were monitored. This study was approved by Experimental Animal Ethics Committee of Zhanjiang Central People's Hospital (ZJDY2023-53). 2.4. Echocardiography and histological analysis Transthoracic echocardiography was performed at 8, 12, and 16 weeks to assess cardiac structure and function. Echocardiographic parameters were measured and averaged over 3 consecutive cardiac cycles. Left ventricular end-systolic volume and left ventricular ejection fraction were measured by M-mode echocardiography. Based on in vivo echocardiographic studies of heart function, the hearts of each group were collected, weighed and the ratio to tibial length was calculated. Major organs such as the heart are fixed with a 10 % formalin buffer, embedded in paraffin, and cut into 5 μm thick sections. Hematoxylin-eosin staining (H&E), Masson staining, Wheat germ thrombin (WGA) staining, and Oil red O staining were used to observe the changes of myocardial pathology. 2.5. Fasting and random blood glucose levels and glucose tolerance tests Fasting and random blood glucose levels were investigated at 8, 12, and 16 weeks. 16-week-old mice were subjected to insulin tolerance test (ITT) (0.5 U/kg) (n = 6) and intraperitoneal glucose tolerance test (IPGTT) (1 mg/g), respectively, and the area under the curve (AUC) was calculated. 2.6. TEM was used to detect the ultrastructural changes of myocardial tissue The tissues were washed in phosphate buffered saline (PBS) and fixed in 2.5 % glutaraldehyde, then fixed with 1 % osmium tetroxide buffer, dehydrated with graded ethanol series and embedded in 812 resin. Ultrathin sections were stained with uranyl acetate water and lead citrate. The image was taken at JEM-100CX-II TEM (Joel, Japan) at 80 kV. 2.7. Culture and treatment of cardiomyocytes To simulate the pathological environment of diabetes, H9C2 cardiomyocytes (ATCC) were subjected to normal glucose (5.5 mmol/L, NG) or high glucose (33 mmol/L, HG) conditions for 48 h. To test the potential therapeutic effect of Emp on H9C2 cardiomyocytes, HG medium was supplemented with 5 μM Emp (Selleckchem, USA). 2.8. Apoptosis assay Apoptosis in cardiac tissues was analyzed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit (C1090, Beyotime, China), using the procedure according to the manufacturer's instructions. The apoptosis rate of H9C2 cells was analyzed by flow cytometry using the annexin V-APC/PI apoptosis kit (E-CK-A217, Elabscience, China) following the manufacturer's instructions. 2.9. Mitochondrial isolation Cardiomyocytes were isolated from mouse heart tissue by centrifugal precipitation. The cells were centrifuged at 1200 r/min for 5 min, the supernatant was discarded, and the cell precipitate was resuspended with 5 % FCS/DMEM medium and incubated in an incubator; after 90 min of cell apposition, the suspension of unapproximated cardiomyocytes was aspirated as described previously [[113]24]. The mitochondria were isolated from cardiomyocytes using a mitochondrial isolation kit (C3601, Beyotime, China). The total mitochondrial protein concentration was determined by BCA protein assay kit (P0010S, Beyotime, China). 2.10. Enzyme activities of HMGCS2, OXCT1and BDH1 Mouse hearts were collected and immediately assayed for enzyme activity. HMGCS2, OXCT1 and BDH1 enzyme activities were measured as previously described [[114]13,[115]25,[116]26]. Enzyme activities were expressed as mmol/min/g protein. 2.11. β-OHB and AcAc concentrations β-OHB levels in mouse frozen heart tissue, serum and urine were determined using a β-hydroxybutyric acid colourimetric kit (JL-T1392, JianglaiBio, China) according to the manufacturer's instructions. AcAc levels in myocardial tissue and serum were determined using a mouse acetoacetate enzyme-linked immunosorbent assay (ELISA) kit (2M-KMLJM220724 m, Camilo, China). 2.12. Mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) ratio Total DNA from mouse hearts was extracted using the DNeasy Blood and Tissue Kit (69504, QIAGEN, Germany). mtDNA and nuclear DNA copy numbers were quantified by real-time qPCR of cytochrome b and β-actin, respectively, as described previously [[117]27]. 2.13. Mitochondrial electron transport chain (ETC) analysis An ELISA microplate assay kit (M150924-8, Mreda, China) was used to measure mitochondrial respiratory chain complex activity according to the manufacturer's instructions. Results are expressed as relative activity (specific respiratory chain complex activity/citrate synthase activity). 2.14. Cardiomyocyte mitochondrial reactive oxygen species (ROS), ATP and oxidative markers assessment MitoSOX Red staining ([118]M36008, Invitrogen, USA) was used to assess mitochondrial ROS levels in cardiac tissues. Mitochondrial ROS was detected in H9C2 cells using a ROS assay kit (S0033, Beyotime, China), and then detected using flow cytometry according to the protocol provided by the manufacturer. ATP concentration in the cardiomyocytes was measured using an ATP assay kit (S0026, Beyotime, China), following the manufacturer's instructions. The results of each assay were normalized by protein concentration as described previously [[119]28]. The results were shown as mmol/mg protein. The levels and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), Catalase (CAT) and malondialdehyde (MDA) were measured according to the ELISA kit instructions to further assess the level of oxidative stress (2 R-KMLJr30259, 2M-KMLJM220779 m, 2M-KMLJM220658 m, 2M-KMLJM219464 m, Camilo, China). The levels of 4-hydroxy-2-nonenal (4-HNE), 3-nitrotyrosine (3-NT), and protein carbonyl in myocardial tissues were determined using an ELISA assay kit ( CSB-E13411-13 m, Cusabio, China), and were expressed in nmol/mg protein as measured according to the manufacturer's protocol. 2.15. Immunofluorescence staining Myocardium sections or cell suspensions were inoculated in petri dishes, fixed with pre-cooled 4 % paraformaldehyde, and rinsed with PBS 3 times for 5 min each time. Block serum (5%FBS+0.01 % Triton X-100 dissolved in PBS) was added and incubated at 37 °C for 60 min. After sealing, diluted Nrf2 (1:50, Abcam) was added to a wet box at 4 °C overnight, and rinsed with PBS 3 times for 5 min each time. Red fluorescent secondary antibody (1:200) was added away from light, incubated at 37 °C for 30 min, and rinsed 3 times with PBS for 5 min each time. The anti-fluorescence quenching sealing tablets containing 4′, 6-diamidino-2-phenylindole (DAPI) were observed under fluorescence microscope. 2.16. qRT-PCR Total RNA was extracted by TRIzol method. The purity and concentration of RNA were determined, and then reverse-transcribed into cDNA, and qRT-PCR was performed by StepOne Plus real-time fluorescence quantitative PCR. The primers were synthesized by GenePharma ([120]Table S1). Data were processed using 2^−ΔΔCT method. 2.17. Western blot It was used to detect key enzymes of target ketone bodies, mitochondrial markers, apoptosis and main regulatory factors of antioxidant stress protein levels. Type-specific antibodies were shown in [121]Table S2. The protein was extracted by adding cell lysate and protease inhibitor. Protein samples were taken for electrophoresis. After electrophoresis, the film was transferred, 5 % skim milk powder was enclosed at room temperature for 1 h, then antigen and antibody reaction were performed, and the target bands of the images were exposed in a dark room according to the instructions of the enhanced chemiluminescence (ECL) luminescence kit (P0018 M, Beyotime, China). Finally, the Image J software was used for gray analysis. 2.18. Immunohistochemistry (IHC) The expression levels of key ketone metabolizing enzyme protein in the heart of each group were detected by IHC. Paraffin sections of mouse hearts were routinely dewaxed to water, antigen repair, PBS washing, added to the configured primary antibody ([122]Table S2), and overnight on a rocking bed at 4 °C; After repeated washing with PBS, adding secondary antibody, diaminobenzidine (DAB) color development, gradient dehydration with ethanol, xylene transparent, neutral resin seal, observation under microscope. 2.19. Statistical analysis Statistical analysis was performed using GraphPad Prism 9.0 and R software. Experimental data are expressed as mean ± standard deviation (SD). Comparisons among multiple groups were made by a one-way analysis of variance (ANOVA), followed by a Tukey post hoc analysis to determine statistical significance. Values of P < 0.05 was considered to be statistically significant. 3. Results 3.1. Identification of DEGs in diebetic hearts By utilizing the high-throughput Gene Expression GEO database combined analysis of [123]GSE215979, [124]GSE161931, [125]GSE145294, [126]GSE161052, [127]GSE173384, [128]GSE123975, [129]GSE161827, [130]GSE210612 and [131]GSE5606. The 9 DCM expression datasets, totaling 38 pairs of samples, are shown in [132]Fig. 1A for a simple flow chart. R-package RobustRankAggreg was used for final expression analysis of 9 different chips or high-throughput sequencing, and the adjusted P value < 0.05 was the standard. 259 DEGs were obtained, among which 226 genes were up-regulated and 33 mRNA were down-regulated. the heatmap for the top 20 DEGs is displayed in [133]Fig. 1B, in addition, Heat maps show the top 80 DEGs using the R package RobustRankAggreg in [134]Fig. S1. Volcanoes were mapped using ggplot2 to show up-regulated and down-regulated genes in 9 datasets respectively ([135]Fig. 1C). Using the adjusted P value < 0.05 and Log2FC ≥ 1 as the criteria, no differential genes were obtained in the [136]GSE145294 and [137]GSE210612 datasets, while 404 up-regulated genes and 464 down-regulated genes were obtained in the other 7 datasets. The results were visualized using UpSetR package ([138]Fig. 1D). Among the up-regulated genes, "[139]GSE215979″, "[140]GSE123975″, "[141]GSE161052″, "[142]GSE161827″, "[143]GSE173384″ and "[144]GSE5606″ were intersected to obtain HMGCS2 and acyl-CoA thioesterase 1 (ACOT1) overlapping genes. Among the down-regulated genes, "[145]GSE215979″, "[146]GSE123975″, "[147]GSE173384″ and "[148]GSE5606″ share a GCAT overlapping gene. The heat map shows that the top 10 differential genes up-regulated and down-regulated are shown in [149]Fig. 2A, and the summary heat map of DEGs is shown in [150]Fig. 2B. Fig. 1. [151]Fig. 1 [152]Open in a new tab Identification of DEGs and overlapping DEGs in the Diabetic or non- Diabetic heart. (A) Experimental flowchart. Gene expression profiles from the GEO public dataset were analyzed to identify DEGs in cardiac tissue. (B) log2FC heat maps representing the image data of each microarray. Note: The horizontal coordinate is GEO ID, and the vertical coordinate is the name of the gene. Blue indicates log2FC > 1, red indicates log2FC < 1 (except [153]GSE145294). (C) Volcano map of differentially expressed RNA in each gene chip. (D) UpSetR plot with 9 data sets overlapping homologous DEGs. (For interpretation of the references to colour in this figure legend, the reader is referred to