Given the typical modern-day high-calorie diet and sedentary lifestyle, obesity has become a worldwide epidemic [1
]. Clinical and epidemiological evidence has shown that obesity is the predominant risk factor for cardiovascular disease (CVD), particularly coronary heart disease, arrhythmias, hypertension, heart failure, and sudden cardiac death [3
]. Obesity is a contributor to increased morbidity and mortality, primarily from CVD. Furthermore, obesity contributes to the development of heart failure by 33% in a BMI-dependent increment [4
]. Excess body weight and accumulated abdominal fat result in cardiac hemodynamic alterations. Consequently, such alterations lead to an adverse cardiac structure and ventricular function, especially an abnormal left ventricular geometry and diastolic capacity [3
]. Obesity-mediated hemodynamics in conjunction with metabolic alterations and neurohormonal abnormalities are the primary contributors to accelerated cardiac remodeling outcomes [5
]. In particular, growing evidence suggests that metabolic alterations occurring in obesity are causative in cardiac remodeling [6
To support the high energetic requirement during cardiac muscle contractions, the cardiac metabolic network utilizes multiple substrates for ATP production, including glucose, fatty acids, and amino acids [8
]. Obesity causes cardiac metabolism changes resulting in the inhibition of the metabolic network responsible for glucose utilization and oxidation, making ATP production and utilization less efficient [10
]. Impairment in coordinated signaling pathways has been observed in essential cardiac metabolic genes via allosterism and post-translational protein modifications. Additionally, the cardiac metabolism network is generally modulated by metabolic regulators, particularly transcription factors [11
]. Although previous studies revealed that a high-fat diet (HFD) induced significant alterations in the metabolic profile that are closely related to cardiac remodeling [13
], detailed information on the influence on cardiac metabolism network alterations and biochemical adaptations occurring in HFD remain unclear. Systems biology approaches have been applied to elucidate the mechanisms of CVD, providing a comprehensive perspective on biological changes [15
]. Transcriptome analysis could provide global information on gene expression profiling in HFD-induced cardiac remodeling, and the mechanism by which HFD influences the cardiac glucose metabolism network, which will serve to develop the current understanding of potential regulatory targets in obesity-induced CVD. Based on the bioinformatic analysis, the core genes in metabolic enzymes, translational factors, and metabolic intermediates could be identified.
Orphan nuclear receptor (Nr4a1
), also known as Nur77, NGFI-B, or TR3, is a transcription factor of the Nr4a subfamily involved in a wide array of important biological processes, including stress response, metabolism, and cell apoptosis [17
]. Nr4a1 has emerged as a therapeutic strategy for suppressing cardiac fibrosis and heart failure via transcriptional regulation of cardiomyocyte apoptosis-related genes [19
]. Another study showed that translocation of Nr4a1 from the nucleus to the mitochondria in neonatal rat cardiomyocytes induced by oxidative stress could also contribute to cardiomyocyte apoptosis [20
]. Recently, its central role in linking CVD to metabolic stress has been identified. Nr4a1
serves as a glucose sensor in the adult heart and may mediate part of glucose’s effects in the stressed myocardium [21
]. It negatively regulates cardiac contractility by regulating the hexosamine biosynthetic pathway and cardiac O-GlcNAcylation [22
]. Additionally, Nr4a1
regulates lipid metabolism in muscle cells by modulating the expression of lipid transporter proteins, such as CD36 [23
]. However, its principal role in modulating myocardial metabolism homeostasis in response to HFD has not been fully illustrated.
In the current study, transcriptome analysis was constructed to obtain whole cardiac transcriptome profiling alterations in response to HFD-induced obesity. With the inclusion of Nr4a1, the primary role of several glucose-homeostasis metabolism-related genes was identified and further validated using in vivo and in vitro tests. The regulatory effect of Nr4a1 in the metabolic adaptation of cardiac remodeling in response to HFD was investigated. Hence, our study provides a comprehensive insight into obesity-related cardiac transcription alterations that will serve to unravel the underlying mechanisms. In addition, a promising role of Nr4a1 was illustrated as a target in the prevention of obesity-induced CVD.
2. Materials and Methods
2.1. Animals and Diets
Twenty-four 8-week-old male C57BL/6J mice were purchased from the Experimental Animal Center of Jilin University (China). All animals were housed in a barrier system under regulated temperature (21±2 °C), humidity (50±5%), and a light-dark cycle of 12 h per day. Before the initiation of the experiments, all mice were acclimatized for 7 days. Mice were randomly divided into a normal diet (ND) group and a high-fat diet (HFD) group of n = 12 mice per group. Mice in the ND group were fed standard chow (12% kcal fat, 23% kcal protein, 65% kcal carbohydrate) for eight weeks. In contrast, mice in the HFD group were fed a high-fat diet (Research Diets D12492 diet, 60% kcal fat, 20% kcal protein, 20% kcal carbohydrate) for eight weeks. Animals had free access to food and water. At the end of experimental period, biological samples were collected after mice were euthanized using carbon dioxide. All animal studies were performed according to the institutional guidelines for the care and use of laboratory animals. Protocols were approved by the Laboratory Animal Ethics & Welfare Committee at the College of Basic Medical Sciences of Jilin University (Approval Number 2018092).
2.2. Biochemical Analyses
Body weight was measured weekly throughout the experiment. Blood glucose was measured at the end of 8 weeks. At the end of experimental period, blood samples of the fasted mice were collected and placed on ice for 30 min. Then centrifuged at 3000 rpm for 10 min at 4 °C to get the serum samples that were kept at −80 °C for biochemical analysis. Serum triglyceride (TG) and total cholesterol (TC) contents were measured enzymatically using commercial kits (Cat#A110-1 and Cat#A111-1, Nanjing Jiancheng Bioeng. Inst.). The activities of lactate dehydrogenase (LDH, Cat#2401131) and creatine kinase (CK, Cat#22401127) were measured by commercial kits obtained from BioSino Bio-Technology & Science Inc. (Beijing, China). Three biological repeats were performed for each sample.
2.3. Heart Histopathology
The cardiac tissue was infiltrated into 10% formalin buffer for 24 h to dehydrate and fix the tissue. Next, the tissue was trimmed and embedded in paraffin. Sections (5 μm thick) were stained with hematoxylin and eosin (H&E) and Sirius Red staining. Circularly polarized images were obtained using Axio Imager M2 for polarized light microscopy (ZEISS, Oberkochen, Germany). H&E staining can display general morphological and structural characteristics in left ventricular myocardium. Quantitative analysis of cardiomyocyte cross-sectional area was conducted by measuring 30 cardiomyocytes from 3 views per section. In Sirius Red staining, the collagen fibers are stained with picrosirius red and viewed with polarized light, depending on the thickness of the collagen fibers. The hues range from green to yellow to orange to red. Cardiac collagen area of left ventricular myocardium was quantitated in ND and HFD groups. The mean area of collagen deposition was obtained by summation of Sirius red-positive areas on each section divided by the total numbers of sections. Image-pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) was applied for quantitative analysis.
2.4. Total RNA Extraction and Validation
Total RNA of whole myocardial tissue was extracted using the mirVana miRNA Isolation Kit (Ambion Inc., Austin, TX, USA) following the manufacturer’s protocol, n = 3 mice per group. RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Samples with an RNA Integrity Number (RIN) ≥ 7 were subjected to subsequent analysis. The libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’ s instructions. These libraries were then sequenced on the Illumina sequencing platform (HiSeqTM 2500 or Illumina HiSeq X Ten), and 125 bp/150 bp paired-end reads were generated.
2.5. Transcriptome Data Mapping and Differential Expression Analysis
Raw data (raw reads) were processed using Trimmomatic software. The reads containing ploy-N and low-quality reads were removed to obtain clean reads. Following this, clean reads were mapped to the reference genome using HISAT2. The FPKM value of each gene was calculated using Cufflinks, and the read counts of each gene were obtained using htseq-count. Differentially expressed genes (DEGs) were identified using the DESeq (2012) R package functions to estimate size factors and the nbinom test. A p
-value < 0.05 and fold change >2 or fold change <0.5 was set as the threshold for significantly differential expression. A hierarchical cluster analysis of DEGs was performed to explore gene expression patterns. Gene ontology (GO) term enrichment [24
] and Kyoto Encyclopedia of Genes and Genomes (KEGG) [26
] pathway enrichment analysis of DEGs were performed using R, based on the hypergeometric distribution.
2.6. H9c2 Culture and Treatment
The embryonic rat heart-derived cell line H9c2 was obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; Gibco, Eggenstein, Germany) containing 2.25 g/L glucose medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 mg/mL of streptomycin, at 37 °C with 5% CO2 in humidified air. Cells between passages 4 and 8 were used for the experiments. For in vitro studies, stock solutions of 5 mM PA/10% fatty acid-free bovine serum albumin (BSA) were prepared and stored at −20 °C. Stock solutions were heated for 5 min at 65 °C and then cooled to room temperature before use. The fatty acid-BSA complex was added to the serum-containing cell culture medium to achieve a fatty acid concentration of 100 μM.
Commercial Nr4a1 siRNA was used to inhibit NR4A1 protein expression according to the manufacturer’s instructions. Transfection of H9c2 cells with siRNAs was carried out using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. H9c2 cells were treated with a mixture containing DMEM, lipo2000, and Nr4a1 siRNA for 4 h. Subsequently, the mixture was replaced with DMEM and cultured for 72 h. Specific siEGFR sequences were as follows: sense, UGGCCCAGAGUUCCCUGAAdTdT, and antisense, UUCAGGGAACUCUGGGCCAdTdT. Transfected cells were then treated with PA, as previously described.
2.7. Quantitative Real-Time PCR (qPCR) Validation
According to the microarray results, the glucose metabolism-related DEGs and key enzymes in glycolysis were chosen for further validation by qPCR in HFD mice and PA-induced H9c2 cardiomyocytes. Cardiac samples were collected, and total RNA was isolated using TRIzol®
reagent (Invitrogen, Carlsbad, CA, USA), and complementary DNA was synthesized using the TransScript
R Frist-Strand cDNA Synthesis SuperMix (TransGen, Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. Primer sets for selected genes were designed by Sangon Biotech (Shanghai, China); their sequences and reaction conditions are listed in Table S1
. Each sample was run in triplicate in 96-well plates using LightCycler R
96 and FastStart Essential DNA Green Master (Roche Diagnostics GmbH, Mannheim, Germany). Quantification cycles were calculated using the fit point method (LightCycler R 96 Software, Version 1.1, provided by Roche). The expression data were normalized to the reference glyceraldehyde-3-phosphate dehydrogenase (GADPH). All experiments (sample collection, preparation and storage, primer design, and qPCR normalization) were performed according to the Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Three biological repeats were performed for each sample.
2.8. Western Blot Analysis
Protein samples were prepared by homogenizing cardiac tissue in radioimmunoprecipitation assay (RIPA) buffer containing proteinase and phosphatase inhibitors (Complete and PhosSTOP; Roche, Basel, Switzerland). Protein extracts from myocardial tissue were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to 0.22 μm polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were then blocked in Tris-buffered saline with 0.1% Tween 20 containing 5% nonfat dry milk for 2 h at room temperature. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies, including Nr4a1 recombinant rabbit monoclonal antibody (Cat#MA5-32647, Thermo Fisher Scientific, 1:1000) and HK2 recombinant rabbit monoclonal antibody (Cat#ab209847, Abcam, 1:1000;). The housekeeping protein GAPDH (Cat#KM9002T, Sungene Biotech, 1:4000) was used as reference protein to normalize the target proteins during western blot analysis. After washing, the membranes were incubated with secondary antibodies antibody IgG (Cat# A0208, Beyotime Biotech, 1:1000) at the appropriate dilutions for 1.5 h at room temperature, and detected using the enhanced chemiluminescence substrate kit. The density of each band was quantified using Quantity One. Three biological repeats were performed for each sample.
2.9. Statistical Analysis
Values represent the mean ± SD (standard deviation). Statistical significance of differences was determined by Student’s t-test or one-way ANOVA with Bonferroni’s post-hoc comparison, where appropriate. Thereby, p-values less than 0.05 were considered significant. SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis.
Obesity has reached epidemic proportions worldwide owing to the paradigm shift to a high-calorie diet and a sedentary lifestyle. Moreover, obesity has placed a heavy financial burden on healthcare costs related to numerous pathologies, especially CVD, including coronary artery disease, arrhythmias, heart failure, and sudden cardiac death [29
]. In the current study, we investigated the global molecular events implicated in the mechanism of cardiac structural remodeling using RNA-Sequencing analysis in an HFD-induced obesity mouse model. The large datasets revealed a global cardiac transcriptome response to HFD challenge; furthermore, valuable genes and pathways implicated in cardiac injuries were identified. In particular, we focused on DEGs involved in glucose metabolism and highlighted the role of NR4A1 in glucose management. These alterations were verified in obesity mouse cardiac tissue and PA-induced H9c2 cardiomyocytes to uncover novel potential targets for the prevention of diet-induced cardiac injuries.
In response to an eight-week HFD, transcriptional profiling data revealed 184 significantly altered genes expressed in cardiac tissues. Functional enrichment analysis revealed that the major biological processes are the cellular response to corticotropin-releasing hormone stimulus, positive regulation of glucose import, and oxygen binding when obesity occurs. These DEGs are mainly implicated in the key components of glucose metabolism, including folate biosynthesis; butanoate metabolism; glycine, serine, and threonine metabolism; and glycolysis/gluconeogenesis metabolism pathways in which were the critical pathways influenced by obesity. Thus, the metabolic dysfunction of myocardium was exacerbated by long-term HFD, especially the dysregulation of glucose metabolism. Obesity causes changes in cardiac glucose metabolism closely connected with the reconstructed energy supply network, which makes ATP production and utilization less efficient. This inefficiency results in functional consequences linked to the increased rate of heart failure in the population [12
]. During energy metabolism remodeling, increased circulating fatty acids and insulin resistance appeared in response to HFD; accompanied by excess fat storage and inhibition of the metabolic machinery responsible for glucose utilization and oxidation [32
]. Consistently, the current cardiac transcriptome analysis revealed dysregulated glucose homeostasis induced by HFD, with Fgf21
, and Hmgcs2
serving as the hub genes. These genes are mainly involved in glucose uptake and glycolysis/gluconeogenesis metabolism modulation processes attributed to myocardial glucose homeostasis.
Significant dysregulated glycolysis related genes were observed in obese mice myocardium, especially Gck
encodes HK4, which catalyzes the phosphorylation of glucose to glucose 6-phosphate (G6P), thereby serving as the first rate-limiting step in glucose metabolism. Compared to other hexokinases, GCK has a weak affinity for D-glucose and is effective only when glucose is abundant [33
]. Generally, GCK is located in the cytoplasm; however, it can shuttle into the nucleus, which may be implicated in gene transcription/new protein synthesis [34
]. Bisphosphoglycerate mutase (BPGM) participates in the production of 2,3-bisphosphoglycerate in glycolysis. Furthermore, BPGM regulates hemoglobin oxygen affinity by controlling its allosteric effector, 2,3-bisphosphoglycerate [35
]. By managing glycolytic intermediate levels, BPGM thereby modulates downstream processing of glycolysis and serine biosynthetic flux [36
]. It has been reported that decreased BPGM was attributed to a failure in the management of glucose homeostasis in acute doxorubicin cardiomyopathy, which may influence the delivery of oxygen to the heart [37
]. Phosphoenolpyruvate carboxylase (PCK) participates in the conversion of oxaloacetate to phosphoenolpyruvate in glycogenolysis. With respect to gluconeogenesis, a particular cellular distribution of PCK has been recently described in the liver and kidney of mice and rats and was also observed to be expressed in myocytes [38
]. Increased expression of PCK1 in the myocardium implicates the promotion of myocardial glycogenolysis by suppressing glycolysis or glycogen synthesis [39
]. Comprehensively, alterations of these hub genes demonstrate the suppression of glycolysis in the myocardium induced by HFD, which is attributed to a disturbance in glucose homeostasis in vivo and in vitro.
Glucose homeostasis is actively orchestrated and carefully choreographed in response to body energy requirements. Maintenance of glucose homeostasis is not limited to the control of metabolic enzymes but is also associated with the regulation of hormones, translational factors, and metabolic intermediates. Glucose transporter-mediated glucose transmembrane uptake serves as the initial step in myocardial glucose metabolism. Slc2a3
, commonly known as Glut3
, is expressed in tissues with heightened energy demands and high metabolic rates since it has the highest glucose affinity and greatest transport capacity in the glucose transporter protein family [40
]. Hence, it is highly expressed in the brain, cardiomyocytes, and cardiomyoblasts [41
]. Decreased expression of Slc2a3
was observed in the cardiac tissue of HFD mice, indicating dysregulated myocardial glucose transport in response to obesity. The regulator of this process, Fgf21
, was also identified as a hub gene in obesity-induced cardiac tissue. It has been demonstrated that FGF21, an endocrine hormone, could facilitate glucose uptake by circulating in peripheral tissues [42
]. Mice lacking FGF21 are more susceptible to the development of obesity-induced cardiac remodeling [43
]. Hepatic-generated FGF21-mediated glucose uptake is dependent on the transcriptional activities of carbohydrate-sensitive transcription factor carbohydrate-responsive element-binding protein (ChREBP) and peroxisome proliferator-activated receptor-α (PPARα), which bind onto the Fgf21
promoter to induce its expression [44
]. The activation of ChREBP was in response to the content of the glycolysis intermediate G6P in the liver; hence, forming a feedback regulation between Fgf21
and glucose metabolism via glycolysis [45
]. Here, we observed significant alterations in Gck
expression, indicating a severely disturbed glycolysis metabolism in the myocardium, which was stimulated by obesity.
Evidence in the current study identified Nr4a1
as the critical regulator of cardiac glucose homeostasis in response to nutrient stimulation. Here, we showed that the expression of cardiac Nr4a1
significantly increased in the myocardium of HFD mice and PA-treated H9c2 cells, which may result in alterations of DEGs in glucose metabolism. However, inhibition of Nr4a1
could enhance glycolysis by enhancing the expression of the key step genes, such as Gck
, and Pkm2
, as well as modulating Fhgf21
mRNA. Consistent with our results, Nr4a1
promotes gluconeogenesis and suppresses glycolysis by attenuating sumoylation-regulated Pck
stability in an acetylation-dependent manner in human hepatoma cell lines [46
]. As a transcription factor, Nr4a1
can bind to the promoter region of the Nur77-binding response element of glycogenolysis and glucose uptake-related genes in muscle, thereby regulating glucose metabolism [47
]. Recently, considerable attention has been paid to its function in the cardiovascular system, but limited mechanisms have been obtained. Cardiac overexpression of Nr4a1
promotes a disturbance in cardiomyocyte calcium homeostasis and adverse cardiac remodeling, depending on the glucose regulation mechanism [22
could negatively regulate cardiac contractility via activation of the hexosamine biosynthetic pathway with downstream effects on protein O-linked glycosylation in the myocardium. As we described, knockdown of Nr4a1
in H9c2 cells resulted in upregulated levels of HK2, suggesting that Nr4a1
may function upstream of HK2, or have an effect on the transcription and translation process of HK2; therefore, handling glycolysis and glucose metabolism homeostasis. These data indicate that Nr4a1
is implicated in cardiac glucose homeostasis, closely linked to the regulation of the hub glucose metabolic genes, serving as a promising target in obesity-induced cardiac impairment. However, specific molecular mechanisms still need further investigation.