The Histone Methyltransferase Mixed Lineage Leukemia (MLL) 3 May Play a Potential Role in Clinical Dilated Cardiomyopathy Ding-Sheng Jiang,1,2,3 Xin Yi,4,5,6 Rui Li,1 Yun-Shu Su,1 Jing Wang,1 Min-Lai Chen,1 Li-Gang Liu,1 Min Hu,1 Cai Cheng,1 Ping Zheng,1 Xue-Hai Zhu,1,2,3 and Xiang Wei1,2,3 1Division of Cardiothoracic and Vascular Surgery, 2Key Laboratory of Organ Transplantation, Ministry of Education, 3Key Laboratory of Organ Transplantation, Ministry of Health, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 4Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, China 5Cardiovascular Research Institute, Wuhan University, Wuhan, China, and 6Hubei Key Laboratory of Cardiology, Wuhan, China Histone modifications play a critical role in the pathological processes of dilated cardiomyopathy (DCM), while the role and expression pattern of histone methyltransferases (HMTs), especially mixed lineage leukemia (MLL) families, in DCM are unclear. To this end, 12 normal and 15 DCM heart samples were included in the present study. A murine cardiac remodeling model was in- duced by transverse aortic constriction (TAC). Real-time polymerase chain reaction was performed to detect the expression levels of MLL families in the mouse and human left ventricles. The mRNA level of MLL3 was significantly increased in the mouse hearts treated with TAC surgery. Compared with normal hearts, higher mRNA and protein level of MLL3 was detected in the DCM hearts, and its expression level was closely associated with left ventricular end diastolic diameter and left ventricular ejection fraction. However, there was no obvious change in the expression levels of other MLL families (MLL, MLL2, MLL4, MLL5, SETD1A and SETD1B) between control and DCM hearts or remodeled mouse hearts. Furthermore, the dimethylated histone H3 lysine 4 (H3K4me2) but not H3K4me3 was significantly increased in the DCM hearts. The protein levels of Smad3, GATA4 and EGR1, which might be regu- lated by MLL3, were remarkably elevated in the DCM hearts. Our hitherto unrecognized findings indicate that MLL3 has a potential role in the pathological processes of DCM by regulating H3K4me2 and the expression of Smad3, GATA4 and EGR1. Online address: http://www.molmed.org doi: 10.2119/molmed.2017.00012 INTRODUCTION control key cellular processes (eg, cell heart chamber dilation, cardiomyocyte Dilated cardiomyopathy (DCM) is cycle, survival, proliferation and dif- death, systolic dysfunction and conduc- characterized by left ventricular enlarge- ferentiation), especially histone modifi- tion abnormalities (2). Loss of H3K27 ment or dilatation and systolic dysfunc- cation, on DCM was largely unknown. methyltransferase EZH2 in cardiac pre- tion, and remarkable progress had been Recently, pioneering work by Nguyen cursors results in cardiac hypertrophy made in understanding its genetic basis et al. demonstrated that cardiac-specific and fibrosis (3). These studies indicate (1). However, the effect of post-transla- DOT1L (an H3K79-specific histone meth- that histone modifications have critical tional modifications of proteins, common yltransferase) deficiency led to DCM-like roles in the pathologic processes of DCM and typical regulatory mechanisms that pathological changes in mice, including and cardiac remodeling. Histone modifications, including meth- ylation, acetylation, phosphorylation and Address correspondence to Ding-Sheng Jiang or Xiang Wei, Division of Cardiothoracic ubiquitination, play a critical role in gene and Vascular Surgery, Key Laboratory of Organ Transplantation, Ministry of Education, transcription regulation (4). Among them, Key Laboratory of Organ Transplantation, Ministry of Health, Tongji Hospital, histone methylation was newly found; it Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang is balanced by methyltransferases and Ave., Wuhan 430030, China. Phone/Fax: 86-27-8366-3394; demethylases, which add or remove E-mail: [email protected], [email protected]. methyl groups from specific lysine or Submitted January 17, 2017; Accepted for Publication August 1, 2017; Published Online arginine. Methylation at different sites has (www.molmed.org) August 9, 2017. diverse biological functions. For exam- ple, methylation at histone H3 lysine 27 (H3K27) is usually associated with gene repression, while di- and trimethylation 196 | JIANG ET AL. | MOL MED 23:196-203, 2017 RESEARCH ARTICLE at histone H3 lysine 4 (H3K4) often ventricle samples of DCM were col- eventually rinsed in deionized water. occur in coding regions of active genes lected from patients undergoing heart Antigen was unmasked by 10 mM (4,5). In mammals, there are at least nine transplantation (18,19). The normal left sodium citrate unmasking solution at H3K4-specific histone methyltransferases ventricle controls were obtained from sub-boiling temperature for 10 min. The (HMTs), including mixed lineage leuke- donor hearts that were not suitable for slides were blocked with 5% normal mia (MLL) families consisting of MLL, transplantation for noncardiac reasons goat serum (ThermoFisher Scientific) MLL2, MLL3, MLL4, MLL5, SETD1A and (18,19). Informed consent was obtained in tris-buffered saline and Tween 20 SETD1B (6–8). The MLL family members, from the families of all subjects. All the for 1 h at room temperature after being each possessing the highly conserved sup- procedures related to human samples treated with 3% hydrogen peroxide pressor of variegation, enhancer of zeste, complied with the principles outlined in for 10 min. Slides were incubated with trithorax (SET) domain responsible for the Declaration of Helsinki. MLL3 primary antibody (1:25 dilution; HMT activity, exist as distinct multipro- Abgent) overnight at 4°C. The next day, tein complexes (namely complex of pro- Cardiac Remodeling Mouse Model the primary antibody was removed, then teins associated with Set1, or COMPASS) Transverse aortic constriction (TAC) incubated with peroxidase-conjugated with several common subunits, including was performed as previously re- secondary antibody (1:2500 dilution; Ash2, Wdr5, Rbbp5 and Dpy30 (9,10). ported to induce a cardiac remodeling Jackson ImmunoResearch Laboratories) MLLs have been credited as key H3K4 mouse model (20). Briefly, male mice for 1 h at room temperature. The DAB mono-, di- and trimethyltransferases (C57BL/6J) 8–10 wks old with body kit was used to develop color, and finally at enhancers or promoters to facilitate weight 24–27 g were anesthetized. The sections were dehydrated and mounted. gene expression (9,10). Previous studies mice were connected to a MouseVent The images were acquired by using an demonstrated that MLLs contribute to Automatic Ventilator (Kent Scientific) Olympus BX53 light microscope. several types of cancers, acute myeloid after endotracheal intubation. Then, leukemia, embryo development, circadian after thoracotomy was performed, the Western Blot rhythm and brown adipocyte differenti- sternum was retracted using a chest re- Western blot was performed as pre- ation (11–17). However, the role of MLLs tractor. After freeing the aortic arch, TAC viously described (20). In brief, total in clinical DCM remains unknown. was performed between the right innom- protein was extracted from human left In the present study, we aimed to in- inate and left carotid artery using 6.0 silk ventricles by using radioimmunopre- vestigate the expression pattern of MLLs suture against a 27-gauge blunt needle, cipitation assay lysis buffer, and protein in human normal and DCM hearts, and which was removed after constriction concentration was quantified by using in mouse hypertrophic hearts. Our re- was completed. Finally, sternal closure Pierce™ BCA Protein Assay Kit (Ther- sults show that there was no significant and skin coverage were performed. A moFisher Scientific). After being dena- change in mRNA expression levels of all similar procedure, but without con- tured, 20 μg protein was separated by MLLs, except MLL3, in mouse hypertro- stricting the aorta, was performed in sodium dodecyl sulfate polyacrylamide phic hearts and in human DCM hearts the sham-operated group. A Vevo-2100 gel electrophoresis, and then transferred compared with the control group. MLL3 High-Resolution Micro-Imaging System to a polyvinylidene fluoride membrane mRNA level was positively correlated (VisualSonics, Toronto, ON, Canada) (Millipore). After being blocked by with left ventricular end diastolic diam- was used to measure cardiac function. 5% skim milk solution, the membrane eter (LVEDD), but negatively correlated was incubated with primary antibody with left ventricular ejection fraction Histological Staining overnight at 4°C, including H3K4me2 (LVEF). Furthermore, H3K4me2 and pro- Human and mouse heart sections (1:1000 dilution; Cell Signaling Technol- tein expression levels of Smad3, GATA4 were stained with hematoxylin and eosin ogies), H3K4me3 (1:1000 dilution; Cell and EGR1, which are downstream of (H&E) for cardiomyocytes cross-sectional Signaling Technologies), GATA4 (1:1000 MLL3, were significantly elevated in area analysis, and picrosirius red to eval- dilution; Abcam), EGR1 (1:1000 dilution; human DCM hearts compared with nor- uate collagen deposition. These two pro- Cell Signaling Technologies), Smad3 mal hearts. cedures were performed as previously (1:1000 dilution; Cell Signaling Technol- described (21–24). ogies) or GAPDH (1:1000 dilution; Cell MATERIALS AND METHODS Signaling Technologies). The protein