Role Of Gata Transcription Factors In Cardiac Energy Metabolism And Cell Proliferation

Background and objectives:Heart has a tremendous capacity for ATP generation to meet the high demand for energy. In embryonic period and cardiac hypertrophy, heart depends on glucose and lactose as the substrates to produce ATP, and after birth and adult heart are dependent on fatty acids as the substrates for energy requirement. As myocardial lipid and glucose storage ability is limited, substrate selection and ATP generation are controlled by rapid adjustment to quickly adapt to energy requirements of the heart.The adjustment process involves successively expression and interaction of multiple genes at different time points, which form an ordered network. The regulatory network is composed of a series of extremely accurate molecular events in time and space. In the process, even a tiny error will lead to the occurrence of myocardial energy metabolism disorder, such as decreased activity of mitochondrial fatty acid oxidation reduces ATP production. It results in cardiac pump function, systolic function and calcium transport function disorders, and at last induces cardiac hypertrophy and heart failure. Therefore, further study of key genes regulatory mechanism in myocardial energy metabolism can help to better understand the molecular mechanisms and pathological basis of cardiovascular diseases at molecular level, and can provide more clues for the prevention and treatment of cardiovascular diseases and new drug designs.Then, what genes are the key genes in myocardial energy metabolism? Glut4is a gene regulating myocardial energy metabolism. Glut4, a member of the glucose transporter protein family, is involved in the maintenance of whole body glucose homeostasis. In general, Glut4expression is tissue-specifically and hormonally regulated. Glut4null mice exhibit cardiac hypertrophy. In view of this, Glut4as a key gene of myocardial energy metabolism, its decreased or increased expression has important physiological or pathological significance for the regulation of myocardial energy metabolism. To research its regulatory mechanism is an important issue in the field of cardiovascular molecular biology.So, which transcription factors are involved in myocardial energy metabolism? The peroxisome proliferator activated receptors (PPARs) serve as critical regulators of fatty acid transport and P-oxidation, which exist in three isoforms:PPARa, PPARy and PPARδ/β. All three isoforms are expressed in heart. The expression of PPARa and PPARδ/β in heart is more abundant than PPARy. Previous evidence shows that mice with cardiac-specific overexpression of PPARa exhibit upregulation of genes involed in fatty acid uptake and β-oxidation. Cardiomyocyte-restricted PPARδ/β deletion in mice impairs myocardial fatty acid oxidation, leading to progressive myocardial lipid accumulation and causes lipotoxicity. Therefore, PPARs play an important role in regulating cardiac energy metabolism.The GATA family of transcription factors comprises six members, and all contain two zinc fingers and bind to the sequence (A/T)GATA(A/G). In the family, GATA4and GATA6are becoming focus in the field of cardiovascular research. A large number of studies have shown that GATA4and GATA6are important transcription factors in regulating heart development and myocardial cell proliferation, differentiation and apoptosis. However, it is not yet clear whether GATA4and GATA6regulate cardiac energy metabolism.MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNAs that regulate target gene expression by post-transcriptional mechanism. Recent evidence has revealed that miRNAs is an important component of cardiac gene transcription regulatory network. Little has been reported whether miRNAs target GATA proteins and PPARs and then influence cell phenotype.Therefore, by using a variety of methods, we studied the regulation mechanism of GATA6, PPARa, GATA4, and miR-200b on myocardial energy metabolism and cell proliferation.Methods and results: Part I Transcription factor GATA6recruits PPARa to cooperatively activate Glut4gene expression.To further investigate the role of the GATA family in regulation of myocardial energy metabolism, by using transient transfection, Q-PCR, luciferase reporter gene, Western Blot, CoIP, GST pull-down and ChIP assay, we finally confirm transcription factor GATA6recruits PPARa to the GATA binding element of the Glut4promoter and cooperatively activate Glut4gene expression, which is accompanied by the promotion of mitochondrial function and glucose utilization.Using Western Blot and Q-PCR assays, we found that GATA6, PPARa and the PPARa agonist fenofibrate all promote Glut4gene expression, and the coexpression of PPARa and GATA6results in greater activation, suggesting that GATA6and PPARa cooperatively activate the Glut4gene transcription. Co-IP experiment proves that GATA6interacts with PPARa. GST pull-down experiments show that the interaction between GATA6and PPARa mediate by the C-terminal zinc finger of GATA6and N-terminal TAD and zinc finger of PPARa. ChIP and luciferase reporter gene assay further confirm that GATA6recruits PPARa through interacting with its N--terminal TAD and zinc finger to Glut4gene promoter and cooperatively activate Glut4gene expression. Moreover, it is accompanied by the promotion of mitochondrial function and glucose utilization.Our study suggests that transcription factor GATA6is an important regulator of myocardial energy metabolism and nuclear receptor PPARa is a novel transcriptional partner for GATA6to regulate gene transcription process. It provides a new molecular basis to understand the role of GATA6in heart development and heart disease.Part II Effects of doxorubicin and fenofibrate on the activities of NADH oxidase and citrate synthase in mice.Dox is an effective anticancer drug, but its clinical use is limited by its serious toxicity. Mitochondrial dysfunction is one of its potential toxic mechanisms, but its precise mechanisms remain unknown. In Part I, we observed PPARa could protect cells mitochondrial function, so we further studied whether PPARa agonist fenofibrate could protect mitochondrial function and reverse the toxicity of Dox in vivo.Eight-week-old mice were randomly divided into four groups:Group1:vehicle control; Group2:fenofibrate treated; Group3:Dox treated; Group4:fenofibrate and Dox treated. Fenofibrate was administered at a dose of100mg/kg/day for14days via oral gavage, and Dox was intraperitoneally administered at15mg/kg/day on days12,13and14. The mice were sacrificed after14days and mitochondria were isolated from the ventricles, atria, livers, kidneys, lungs and spleens. Then, we tested citrate synthase and NADH oxidase activity. Both enzymes are marker enzymes of mitochondrial function. The results showed that fenofibrate induces the activities of citrate synthase and NADH oxidase in many tissues, whereas Dox inhibit both enzymes activities in most tissues. Furthermore, fenofibrate can reverse the Dox-induced toxicity in ventricle and kidney.The study demonstrates that fenofibrate has a protective effect on mitochondrial citrate synthase and NADH oxidase, and can reverse Dox-induced toxicity in ventricular and kidney. It provides theory basis for how to reduce the side effects of Dox.Part III miR-200b targets transcription factor GATA4to regulate cell proliferation, cell cycle and apoptosisIn order to find the upstream regulators of the GATA family, we found that GATA4may be a target gene of miR-200b by bioinformatic analysis. Evidence shows that miR-200b participates in the regulation of epithelial to mesenchymal transition (EMT) process and it is an important regulator for the formation of tumor cells. Because miRNAs regulate cell proliferation and differentiation and apoptosis, we used stably transfection, RNAi, luciferase reporter genes, MTT, flow cytometry, RT-qPCR and DAPI staining assays to study the role of miR-200b on cell proliferation, cell cycle and apoptosis.The result shows that overexpression of GATA4or miR-200b promotes or inhibits the proliferation of C2C12and P19cells, respectively. Overexpression of miR-200b or knockdown of GATA4by siRNA induced cell GO/G1arrest, and S phase and G2/M phase cells reduced. Moreover, DAPI staining confirmed that miR-200b induced cell apoptosis. Luciferase reporter gene experiments demonstrated that miR-200b directly binded the predicted target site within GATA43'-UTR, and Western Blot further validated miR-200b could inhibit GATA4expression at the protein level, which suggest that GATA4is really the target gene of miR-200b.In this study we prove that GATA4is the target gene of miR-200b. miR-200b regulates the protein expression of GATA4by post-transcriptional mechanism, and then inhibits cell proliferation, triggers cell G0/G1arrest and induces cell apoptosis. These results indicate that miR-200b may regulate heart development, cardiac cell proliferation and apoptosis by targeting GATA4.Conclusions:In the present study, we found:(1) in cardiacmyocytes stimulated by fenofibrate, a PPARa agonist, transcription factor GATA6recruits PPARa to the GATA binding element of the Glut4promoter and cooperatively activate Glut4gene expression, which is accompanied by the promotion of mitochondrial function and glucose utilization;(2) fenofibrate, a PPARa agonist, has a protective effect of mitochondrial citrate synthase and NADH oxidase. Morover, fenofibrate can alleviate doxorubicin (Dox)-induced toxicity in ventricle and kidney;(3) miR-200b targets transcription factor GATA4to inhibit the proliferation of C2C12cells and P19cells, and trigger cell G1arrest and induce cell apoptosis. In conclusion, our research suggests that GATA6is an important component of gene regulatory network in cardiac energy metabolism, and GATA6serves as a co-activator of PPARa to cooperatively regulate cardiac energy metabolism. It provides a new molecular basis to understand the role of GATA6in heart development and heart disease. In addition, our result shows that miR-200b is involved in cell proliferation and apoptosis, which could pave the way for further comprehend the role of GATA family of transcription factors in the heart.