EGCG (>98% purity) was obtained from School of Pharmaceutical Science, Kunming Medical University. Male Sprague–Dawley (SD) rats (SPF, 6 weeks old, 200 ± 20 g) were obtained from the Animal Research Center of Kunming Medical University. Animals were maintained at a controlled temperature (24 ± 2°C) and humidity (60 ± 10%) with a 12 h light/12 h dark cycle. After two weeks of adaptive breeding, SD rats were randomly divided into three groups: a) the control group (n = 10), with a normal diet for 36 weeks; b) HFD-fed group (n = 10), with the HFD (15% lard, 30% sucrose, 2% cholesterol, 1% sodium cholate, 5% protein powder, and 47% regular diet), in which sucrose can be converted into energy and fat, for 36 weeks; c) EGCG-treated HFD-fed group (n = 10), with the HFD for 36 weeks and intragastrically administered with 160 mg/kg/d EGCG for the last 4 weeks (Figure 1B). According to previous studies, 100–200 mg/kg/d EGCG treatment could provide therapeutic effects on early aged hypertension-induced neural apoptosis (20) and senescence-mediated redox imbalance in the livers and kidneys in mice (21). Therefore, we chose 160 mg/kg/d as our experimental dose in our study. This study was approved by the Institutional Review Boards of Kunming Medical University.

Peripheral blood samples were centrifuged at 4,000 g for 10 min at 4°C, and the cleared supernatants were collected. Total cholesterol (TC) and triglycerides (TG) in the serum were determined by using a commercial kit (Leagene Biotechnology, Beijing, China). The concentration of creatine phosphokinase-MB (CKMB) in the serum was determined by an ELISA kit (Xinyu Biotechnology, Shanghai, China). All operations were carried out according to the manufacturer's instructions.

A segment of the heart tissue was excised, fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Serial 5-μm-thick sections were mounted and stained with hematoxylin and eosin (HE) and Masson's trichrome to examine the pathology of the heart. Furthermore, immune histology staining was performed for collagen I (1:200, AF7001, Affinity Biosciences, USA) and collagen III (1:200, AF0136, Affinity Biosciences, USA) in heart sections. Quantitative assessment of the fibrosis was determined based upon the extent of patchy and interstitial fibrosis. Each heart was observed, and at least 5 pictures were obtained in each region.

Total RNA was extracted from the hearts with RNAiso plus (TaKaRa Biotechnology, Beijing, China). The mRNA was reverse transcribed into cDNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Quantitative real-time PCR was performed using SYBR green PCR master mix (Roche, Penzberg, Germany) for 40 cycles in an ABI Q6 apparatus (Applied Biosystems, CA, USA). Real-time fluorescence quantitative PCR was performed using the following primers: Gapdh forwards primer, 5′-TGGCACCACACCTTCTACAATG-3′; Gapdh reverse primer, 5′-TCATCTTCTCGCGGTTGGC-3′; Scn5a forwards primer, 5′-GAAGTGCCAGCGTAACTCCTA-3′; and Scn5a reverse primer, 5′-TACCTCAAACACCCCATTCAC-3′.

Three hearts from each group were subjected to RNA-seq. RNA libraries were generated using Illumina's TruSeq Stranded Total RNA Sample Prep Kit. RNA libraries were sequenced using paired-end 150 bp reads on a NovaSeq system (Illumina, San Diego, CA, USA). Raw read data were subjected to the quality control using FastQC, and unqualified reads were cleaned using fastp (22). The filtered read data were aligned to the Rnor6.0 genome (release 100 in Ensemble) by using HISAT2 (23). Differential expression analysis was performed using DESeq2 (24) and the protein–protein interaction (PPI) network was predicted using the STRING online database (http://string-db.org). Firstly, the gene list was typed into the database. Then, PPI was constructed with the default parameters and displayed with CytoScape (25). Further, the hub genes in the PPI networks were identified using Cytohubba (26), a plugin in Cytoscape software, and the top nodes were ranked by degree.

Total protein from the heart was extracted with ice-cold 1% Triton X-100 RIPA lysate supplemented with PMSF and then quantified by BCA. The protein was loaded in lanes, separated by SDS–PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated with collagen I (1:1000, AF7001, Affinity Biosciences, USA), collagen III (1:1000, AF0136, Affinity Biosciences, USA), Scn5a antibody (1:1000, AF0255, Affinity Biosciences, USA) and GAPDH antibody (1:1000, D16H11, Cell Signaling Technology, USA) overnight at 4°C. Then, the membrane was incubated with HRP-labeled antibodies and exposed to HRP chemiluminescence. ImageJ 1.52 was used to quantify the bands, and GAPDH was used as a loading control to calculate the relative quantification among each group.

Data were presented as the mean and standard error of the mean (mean ± SEM). The Kolmogorov–Smirnov test showed that the data were normally distributed. Differences were evaluated by one-way ANOVA for multigroup comparisons and SNK-Q test for pairwise comparisons. Statistical analysis was performed using SPSS 21.0 and GraphPad Prism 8.0 statistical software. Statistical significance level was set at α = 0.05.

After 32 weeks of HFD and normal diet, the weight of HFD-fed group was significantly higher than that of the control group. (Further, compared with the HFD-fed group, the weight of the EGCG-treated HFD-fed group decreased significantly after 4 weeks of EGCG treatment (P < 0.0001, One-way ANOVA and SNK-Q test, Figure 1C). These results showed that HFD-feeding significantly induced the obesity and EGCG treatment could significantly reduce the obesity in rats. At this point, the rats were euthanized for further experimental evaluation. Heart weight index was an indicator of cardiac hypertrophy measured by heart weight to body weight (HW/BW) ratio. However, no change was found in the heart weight index (P > 0.05, one-way ANOVA test, Figure 1D). Furthermore, the TC and TG levels in the serum were examined and the results showed that HFD feeding could increase the level of blood lipids, while EGCG treatment could significantly decrease the HFD feeding-induced increase in the level of blood lipids (P < 0.0001, one-way ANOVA and SNK-Q test, Figures 1E,F). This result indicated that EGCG could reduce obesity-induced discordance of the lipid metabolism. Discord lipid metabolism could place a severe burden on the heart, so we measured CKMB in the serum to assess the myocardial injury and found that serum CKMB concentration was highest in the HFD-Fed group and lowest in the control group (P < 0.0001, one-way ANOVA and SNK-Q test, Figure 1G). The results showed that HFD feeding aggravated the myocardial injury and EGCG effectively protected against obesity-induced myocardial injury.

Frequent injury and repair of the myocardium can lead to the formation of fibrosis. To investigate the effect of EGCG on the pathological changes in HFD-fed rats, heart tissues were stained with HE and Masson's trichrome separately. HE staining results showed that the tissues in the control group were evenly distributed without obvious inflammatory cell infiltration, while tissues in the HFD-fed group revealed pyknosis, blurred cell boundaries, inflammatory cell infiltration and connective tissue hyperplasia. After EGCG treatment, the infiltration of inflammatory cells was significantly reduced, nuclear pyknosis and connective tissue hyperplasia were significantly improved, the pathological change was rescued. The Masson staining results confirmed the presence of extensive myocardial fibrosis in the HFD-fed group compared to the control group, while the level of myocardial fibrosis in the EGCG-treated HFD-fed group was significantly reduced (Figure 2A).

Furthermore, the accumulation levels of collagen I and collagen III in hearts were analyzed. As shown by immunohistochemistry and western blot analysis, HFD can significantly induce the expression and obvious deposition of collagen I and collagen III in ECM. However, deposition (P < 0.0001, one-way ANOVA and SNK-Q test, Figures 2B,C) and expression (P < 0.0001, one-way ANOVA and SNK-Q test, Figure 2D) of collagen I and collagen III were significantly inhibited by EGCG treatment in HFD-fed rats.

RNA-seq was performed to explore the potential mechanism of EGCG in the treatment of obesity-induced myocardial fibrosis. The read counts were normalized by using DESeq2 before analysis. The principal component analysis (PCA) of the transcriptome data was performed first. The first principal component from this analysis accounted for 35% of total transcriptome variance, it completely separated the control group from other groups. The second principal component from this analysis accounted for 15.9% of total transcriptome variance, it completely separated the HFD-fed group from other groups. The result from PCA revealed the different gene expression value induced by HFD-fed and the treatment of EGCG (Figure 3A). The PCA results revealed the different gene expression profiles induced by HFD feeding and EGCG treatment in hearts.

Then, we identified differentially expressed genes by DESeq2 based on strict statistical criteria (log₂Fold_change > 1.2 and adjusted P value < 0.05). A total of 673 differentially expressed genes, including 275 upregulated and 398 downregulated genes, were identified in the comparison of the control group and HFD-fed group (Supplementary Table 1). A total of 1049 differentially expressed genes, including 738 upregulated genes and 311 downregulated genes, were identified in comparison with the EGCG-treated HFD-fed group and the HFD-fed group (Supplementary Table 2, Figure 3B). This result indicated that both HFD-fed and EGCG treatment resulted in the significant changes in the transcriptome.

To explore the mechanism by which EGCG alleviates obesity-induced myocardial fibrosis, some genes that were changed by a HFD diet and reversed by EGCG treatment were selected for further analysis. First, the intersection of the two analyses of differentially expressed genes was taken to establish a new gene set (Figure 3B). Then, the change direction of the genes in this new gene set, including 375 genes was analyzed. Interestingly, it was found that all the gene expression changes caused by the HFD diet could be reversed by EGCG treatment (Figure 3C). In the end, a total of 375 genes were included in the further analysis. To understand the function of these genes, we enriched these genes with the KEGG database and found that these genes were mainly involved in the pathways related to the heart disease, glycolipid metabolism and calcium signaling, suggesting that EGCG treatment could alleviate the obesity-induced cardiac pathological changes via redistribution of ions or equilibrium of glycolipid metabolism (Figure 3D). A PPI network of these genes was constructed and the core of the network was found to be the sodium-calcium channel, including Scn5a, Ryr2, Hcn4, Cacna2d1, Camk2a, Ank2, Kcnh2 and Cacnb2. The Scn5a gene had the highest degree score of 26 (Figure 3E). Therefore, the expression of Scn5a gene may play a central role in EGCG in alleviating HFD-fed induced myocardial fibrosis.

The above bioinformatics analysis showed that EGCG could alleviate obesity-induced myocardial fibrosis by regulating the expression of Scn5a. Previous studies showed that Scn5a knockout in mice accelerates the progression of myocardial fibrosis (19). The quantitative real-time PCR and western blotting conducted with SCN5A showed that HFD-fed could significantly reduce the expression level of SCN5A, which may be the main reason for the occurrence of myocardial fibrosis. While EGCG treatment could significantly increase the expression of SCN5A both at mRNA level and protein level, it could also reverse the decreased expression level of SCN5A induced by HFD-Fed (P < 0.0001, one-way ANOVA and SNK-Q test, Figures 3F,G).