Short-Chain Enoyl-CoA Hydratase Mediates Histone Crotonylation and Contributes to Cardiac Homeostasis
2021; Lippincott Williams & Wilkins; Volume: 143; Issue: 10 Linguagem: Inglês
10.1161/circulationaha.120.049438
ISSN1524-4539
AutoresXiaoqiang Tang, Xiaofeng Chen, Xin Sun, Peng Xu, Xiang Zhao, Ying Tong, Xiaoman Wang, K. G. Yang, Yutong Zhu, De‐Long Hao, Zhu‐Qin Zhang, De‐Pei Liu, Hou‐Zao Chen,
Tópico(s)Neurological diseases and metabolism
ResumoHomeCirculationVol. 143, No. 10Short-Chain Enoyl-CoA Hydratase Mediates Histone Crotonylation and Contributes to Cardiac Homeostasis Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBShort-Chain Enoyl-CoA Hydratase Mediates Histone Crotonylation and Contributes to Cardiac Homeostasis Xiaoqiang Tang, PhD, Xiao-Feng Chen, PhD, Xin Sun, BSc, Peng Xu, PhD, Xiang Zhao, BSc, Ying Tong, MSc, Xiao-Man Wang, PhD, Ke Yang, PhD, Yu-Tong Zhu, BSc, De-Long Hao, MSc, Zhu-Qin Zhang, PhD, De-Pei Liu, PhD and Hou-Zao Chen, PhD Xiaoqiang TangXiaoqiang Tang https://orcid.org/0000-0003-1314-3417 Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. , Xiao-Feng ChenXiao-Feng Chen Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. , Xin SunXin Sun Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. , Peng XuPeng Xu Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. , Xiang ZhaoXiang Zhao Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. , Ying TongYing Tong Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. , Xiao-Man WangXiao-Man Wang Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. , Ke YangKe Yang Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. , Yu-Tong ZhuYu-Tong Zhu Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. , De-Long HaoDe-Long Hao Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. , Zhu-Qin ZhangZhu-Qin Zhang Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. , De-Pei LiuDe-Pei Liu De-Pei Liu, PhD, State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China. Email E-mail Address: [email protected] https://orcid.org/0000-0002-2636-4297 Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. and Hou-Zao ChenHou-Zao Chen Hou-Zao Chen, PhD, State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China. Email E-mail Address: [email protected] https://orcid.org/0000-0001-6805-3182 Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Originally published8 Mar 2021https://doi.org/10.1161/CIRCULATIONAHA.120.049438Circulation. 2021;143:1066–1069Posttranslational modifications of histones are critically involved in gene expression and regulate pathophysiologic processes such as cardiovascular diseases. Metabolic enzymes modulate the intracellular levels of metabolites to support posttranslational modifications.1 Recently, new histone acylations have been identified to regulate gene expression. For example, histone crotonylation (H3K18cr and H2BK12cr) can trigger gene transcription and regulate metabolism, DNA repair, depression, and reproductive development.2 The roles of histone crotonylation in pathophysiologic processes of cardiovascular diseases—cardiac hypertrophy, for example—remain unknown. Short-chain enoyl–coenzyme A (CoA) hydratase (encoded by ECHS1) is a hydratase that has the highest activity for hydrolyzing crotonyl-CoA, reducing intracellular crotonyl-CoA, the orchestrator of histone crotonylation (Figure [A]).2,3 In human newborns or children, mutations in the ECHS1 gene lead to cardiomyopathies (>60%), such as hypertrophic cardiomyopathy, with unknown mechanisms.3,4Download figureDownload PowerPointFigure. Short-chain enoyl–coenzyme A (CoA) hydratase mediates histone crotonylation and contributes to cardiac homeostasis. A, Schematic diagram shows the metabolism of crotonyl-CoA. Crotonyl-CoA can be used by histone crotonyl-transferase (HCT; eg, P300) to promote histone crotonylation and gene transcription. Histone crotonylation can be reduced by ECHS1 (short-chain enoyl-CoA hydratase), which hydrolyzes crotonyl-CoA to generate short-chain hydroxyacyl-CoA. B, Western blot shows the protein level of ECHS1 in heart tissues from healthy controls (n=6) and human patients with hypertrophic cardiomyopathy (HCM) (n=9). C, Western blot shows levels of crotonylated H3K18 (H3K18cr) and H2BK12cr in heart tissues from controls and patients with HCM. D, Genotype quantification of the offspring of each Echs1 mutant line and analysis of Echs1 expression (n=6 or 7). E, ECHS1 deficiency promotes angiotensin II (Ang II)–induced cardiac remodeling. Wild-type and Echs1 heterozygote mice (8 to 12 weeks old) were subjected to saline or Ang II infusion (1.3 mg/kg/d) for 4 weeks (n=12 to 21). The ratio of heart weight (HW)/body weight (BW) and HW/tibia length (TL) shows that Echs1+/− promotes Ang II–induced increase in HW. Hematoxylin & eosin (H&E) and wheat germ agglutinin (WGA) staining show that Echs1+/− promotes Ang II–induced increase in cardiomyocyte size (n=6 to 10). F, ECHS1 represses Ang II–induced hypertrophy in neonatal rat cardiomyocytes (NRCMs). NRCMs were transfected with siRNA targeting Echs1 (siEchs1) and negative control siRNA (siNC) or infected with adenovirus-carrying control (Ad-Ctrl) and ECHS1-expressing constructs (Ad-ECHS1) for 24 hours. The cells were treated with Ang II (1 μmol/L) for an additional 48 hours. ECHS1 expression was analyzed by Western blot. α-Actinin staining and Image J were applied to analyze cardiomyocyte size (bar, 30 μm). G, Western blot shows the identification of the line of ECHS1-transgenic mice with moderate ECHS1 overexpression. Heart tissues were used for analysis. H, Cardiac ECHS1 transgene expression represses Ang II–induced cardiac remodeling. Wild-type and ECHS1 transgenic mice were subjected to saline or Ang II infusion (1.3 mg·kg−1·d−1) for 4 weeks (n=8 to 18). I, ECHS1 overexpression represses the levels of crotonylated H3K18 (H3K18cr) and H2BK12 (H2BK12cr) in hypertrophic hearts. Heart tissues from panel H were analyzed. J, ECHS1 deficiency promotes histone crotonylation. NRCMs were transfected with siNC or siEchs1, and the cells were treated with the crotonyl-CoA donor (crotonate) at the indicated concentrations for an additional 24 hours. K, Supplement of crotonyl-CoA with its donor crotonate or knockdown of Echs1 induces cardiomyocyte hypertrophy, whereas ECHS1 overexpression represses crotonate-induced hypertrophy in NRCMs. NRCMs with Echs1 knockdown or overexpression along with the controls were treated with crotonate (10 mmol/L) for an additional 48 hours. L, Neonatal rat cardiomyocytes with or without Echs1 knockdown were subjected to bulk RNA sequencing (RNA-Seq). Gene set enrichment analysis shows the enrichment of hypertrophic marker genes in NRCMs with Echs1 deficiency. M, TRANSFAC and JASPAR PWM analysis of upstream transcriptional factors of the upregulated genes (fold change>1.2, P<0.01) by Echs1 knockdown using RNA-Seq data in L (left). Qualitative real-time polymerase chain reaction (qRT-PCR) results show that Echs1 knockdown or crotonate (10 mmol/L) treatment for 48 hours leads to the overexpression of Nppb and Echs1 knockdown enhances the effects of crotonate on Nppb expression (right). N, Chromatin immunoprecipitation assay shows that ECHS1 and crotonate regulate the enrichment of NFATc3 at Nppb promoter. NRCMs with Echs1 knockdown along with the controls were treated with crotonate (10 mmol/L) for an additional 48 hours, followed by chromatin immunoprecipitation and qRT-PCR experiments. O, Summary graph shows the role of ECHS1 and histone crotonylation in regulating cardiac homeostasis. All cell experiments were repeated 3 times and values are expressed as mean±SEM. **P<0.01, ***P<0.001. Mann-Whitney test was applied for the analysis of data in B and D. Two-way analysis of variance with Bonferroni post hoc test was performed to analyze data in E, F, H, K, M, and N. FDR indicates false discovery rate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NFAT, nuclear factor of activated T cells; PBS, phosphate-buffered saline; and TSS, transcription start sites.Downregulation of ECHS1 was observed in human hearts with hypertrophic cardiomyopathy (Figure [B]). ECHS1 downregulation was coupled with the upregulation of H3K18cr and H2BK12cr (Figure [C]), suggesting the involvement of ECHS1 and histone crotonylation in cardiac hypertrophy.To understand the roles of ECHS1 in cardiac hypertrophy, we generated 3 lines of germline Echs1 mutant mice with CRISPR-Cas9. However, no homozygote of Echs1 mutation was obtained, which might be attributable to the embryonic death induced by Echs1 knockout. Echs1 expression in heart tissues of Echs1+/− mice was decreased to ≈50%, and Echs1+/− mice developed normally (Figure [D] and data not shown). Cardiac hypertrophy in male adult Echs1 deficient (Echs1+/−) and littermate wild-type (Echs1+/+) mice was induced by a chronic infusion of angiotensin II (Ang II). Ang II treatment significantly increased heart weight, which was further enhanced in Echs1+/− mice (Figure [E]). Histologic analysis showed that Echs1 deficiency promoted the increase in heart and cardiomyocyte size induced by Ang II (Figure [E]).We also analyzed whether ECHS1 regulated cardiac hypertrophy by directly targeting cardiomyocytes. Echs1 was knocked down with siRNA in neonatal rat cardiomyocytes (NRCMs) and cardiomyocyte hypertrophy was induced by Ang II. Echs1 knockdown alone induced hypertrophic growth and promoted the prohypertrophic effects of Ang II (Figure [F]). To test whether rescuing ECHS1 expression can repress hypertrophic growth of NRCMs, ECHS1 was overexpressed in NRCMs with adenovirus. ECHS1 overexpression repressed Ang II–induced increase in cardiomyocyte size (Figure [F]).To further investigate whether ECHS1 expression in cardiomyocytes can repress cardiac hypertrophy in vivo, we generated 2 lines of mice with cardiomyocyte-specific ECHS1 overexpression. Among them, 1 line moderately expressed ECHS1 and was used for further study (Figure [G]). Ang II–induced increase in heart weight was reduced in ECHS1 transgenic mice compared with the controls (Figure [H]). Consistently, the histochemical analysis revealed that ECHS1 overexpression repressed cardiac hypertrophy (Figure [H]).ECHS1 regulates the level of crotonyl-CoA, which can regulate gene expression by histone crotonylation (Figure [A]).2 H3K18cr and H2BK12cr levels were increased in human hypertrophic cardiomyopathy (Figure [C]). In murine hypertrophic hearts, H3K18cr and H2BK12cr were remarkedly repressed by ECHS1 overexpression (Figure [I]). By contrast, crotonyl-CoA supplement with crotonate increased histone crotonylation in cardiomyocytes, which was enhanced by Echs1 knockdown (Figure [J]), suggesting that ECHS1 repressed histone crotonylation. To test whether histone crotonylation promoted cardiac hypertrophy, cardiomyocytes were treated with crotonate. Crotonate alone increased cardiomyocyte size, which was enhanced by Echs1 knockdown (Figure [K]). By contrast, ECHS1 overexpression resulted in the inhibition of crotonate-induced hypertrophic growth of NRCMs (Figure [K]).Bulk RNA sequencing and gene set enrichment analysis were performed to understand the mechanism by which ECHS1 and histone crotonylation regulated cardiomyocyte hypertrophy. Significantly, Echs1 deficiency led to the enrichment of the hypertrophic cardiomyopathy gene set, and the hypertrophic fetal gene Nppb was enriched in the leading edge of gene set enrichment analysis (Figure [L]). TRANSFAC and JASPAR PWM analysis of transcriptional factors of the ECHS1 downstream genes revealed NFATc3 as the most promising candidate that contributed to the effects of ECHS1 and histone crotonylation (Figure [M], left). NFATc3 is a pivotal transcriptional factor for driving the expression of hypertrophic fetal genes, such as Nppb.5 Indeed, the expression of hypertrophic gene Nppb in NRCMs with Echs1 deficiency was validated (Figure [M], right). Besides, crotonate, which induced histone crotonylation, can lead to the expression of Nppb directly and Echs1 knockdown promoted the effects of crotonate on Nppb expression (Figure [M], right). However, neither ECHS1 nor crotonate affected the expression of NFATc3 in cardiomyocytes (Figure [J]). Instead, chromatin immunoprecipitation assay showed that either crotonate or Echs1 knockdown promoted the enrichment of NFATc3 at Nppb promoter (Figure [N]).Collectively, our study highlights that the hydratase ECHS1 controls the intracellular crotonyl-CoA and maintains the maturity and homeostasis of cardiomyocytes via histone crotonylation and NFATc3 (Figure [O]). Although ECHS1 might also function in a crotonylation-independent manner, our findings partially elucidate the phenotypes and mechanisms underlying ECHS1 mutation-mediated cardiac defects in humans. Therefore, histone crotonylation may serve as a therapeutic target for children with ECHS1 mutations and patients with hypertrophic cardiomyopathy.All protocols using human heart samples were approved by the Ethical Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College. All animal protocols were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College.The data, materials, and methods supporting the findings of this study are available from the corresponding author on request. RNA sequencing data are publicly available with GEO accession number GSE159039.Sources of FundingThis work was supported by grants from the National Key Research and Development Project of China (grant numbers 2019YFA0801500 and 2020YFC2008003), the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (grant numbers CIFMS2017-I2M-1-008 and 2019-RC-HL-006), and the National Natural Science Foundation of China (grant numbers 91849207, 82030017, 81800273, and 81970426).Disclosures None.Footnotes*Drs Tang, Chen, and Sun contributed equally.https://www.ahajournals.org/journal/circDe-Pei Liu, PhD, State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China. Email [email protected]edu.cnHou-Zao Chen, PhD, State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China. Email [email protected]cams.cnReferences1. Gillette TG, Hill JA. Readers, writers, and erasers: chromatin as the whiteboard of heart disease.Circ Res. 2015; 116:1245–1253doi: 10.1161/CIRCRESAHA.116.303630LinkGoogle Scholar2. Liu S, Yu H, Liu Y, Liu X, Zhang Y, Bu C, Yuan S, Chen Z, Xie G, Li Wet al. Chromodomain protein CDYL acts as a crotonyl-CoA hydratase to regulate histone crotonylation and spermatogenesis.Mol Cell. 2017; 67:853–866.e5. doi: 10.1016/j.molcel.2017.07.011CrossrefMedlineGoogle Scholar3. Yamada K, Aiba K, Kitaura Y, Kondo Y, Nomura N, Nakamura Y, Fukushi D, Murayama K, Shimomura Y, Pitt Jet al. 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Jiang M, Xie X, Cao F and Wang Y (2021) Mitochondrial Metabolism in Myocardial Remodeling and Mechanical Unloading: Implications for Ischemic Heart Disease, Frontiers in Cardiovascular Medicine, 10.3389/fcvm.2021.789267, 8 March 9, 2021Vol 143, Issue 10 Advertisement Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.120.049438PMID: 33683949 Originally publishedMarch 8, 2021 Keywordshistonescardiomegalytranscription factorsenoyl-CoA hydratasePDF download Advertisement SubjectsBasic Science ResearchCell Biology/Structural BiologyCell Signaling/Signal TransductionPathophysiology
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