Role Of Methylation Of ATRAP Gene In Regression Of Left Ventricular Hypertrophy In Early Losartan Treated Spontaneously Hypertensive Rats

Background Essential hypertension(EH), characterized by chronic elevation of arterialblood pressure(BP)due to an unknown etiology, is a cardiovascular syndrome. EH has incredible heritability. However, the collective effect of many genetic loci, identified bygenome-wide association studies(GWAS)only accounts for a small fraction of BP heritability in the general population. The reason of the “missing heritability” remains unclear. Data in animal modelsof programmed hypertension have shown that DNA methylation ofepigenetic modification in some genetics mediates the development of hypertension in adulthood resulting from suboptimal enviromental factors in early life.It has been reported that the level of BP in adultspontaneously hypertensive rats(SHR), rat models of genetic hypertension, are also associated with enviromental factors in early life. So it is reasonable to speculate that the epigenetics is one of the important regulators of the pathogenesis and development of EH. Our previous studies have found that shortterm treatment of SHR with angiotensin receptor blocker(ARB) losartan(Los) in early life could persistently postpone the increase in BP, inhibit left ventricular hypertrophy(LVH), alter angiotensin type 1 receptor(AT1R)in myocardium, a key gene of reninangiotensin system(RAS).Angiotensin type 1 receptor-associated protein(ATRAP), as a negative regulator of AT1 R signalling, extensively presents in cardiovascular system. It has been documented that overexpression of ATRAP in myocardium may inhibit the development of LVH and ARB is capable of inhibiting ATRAP expression. On these bases, the aim of this study is to investigate the role of methylation of ATRAP gene in regression of LVH in early losartan treated SHR. The findings of the study are expected to provide an insight into the contribution of epigenetics to “missing heritability”of EH,and warrant further investigations for a novel therapeutic strategy of short term intervention, long-term reduction in BP.Objectives 1. To investigate the long-term influence of early treatment of SHR with Los on BP, LVH and cardiac ATRAP expression in adult. 2. To identify the difference in the DNA methylation of ATRAP gene promoter in myocardium between SHR and normotensive Wista-Kyoto(WKY)rats and to explore the long-termeffect of early treatment with Los on the methylation of ATRAP gene. 3. To examine the effects of Los on the rate of protein synthesis and ATRAP expressionin H9c2 cellsinducedby angiotensin II(Ang II),and to elucidate the role of DNA methylation in the effects of Los.Methods Animal experiment(1) Experimental design: SHR were randomly divided into four groups: untreated SHR group(SHRUn), prenatal Los-treated SHR(SHRPre), postnatal Los-treated SHR(SHRPost) and sequential Los-treated SHR(SHRSeq), and untreated pregnant WKY(WKYUn)served as control group.SHRPre 、SHRPostand SHRSeqgroupswere considered as early treament groups. WKYUn and SHRUn:Both maternal rats and offspring rats were given the treatment without Los; SHRPre: maternal SHR were administrated Los only during the pregnancy; SHRPost: maternal SHR received Los treatment during the lactation, and after weaning, the offspring continued to receive Los treatment until they were 8 weeks old; SHRSeq: maternal SHR were administrated Los throughout the pregnancy and lactation, and after weaning,the offspring continued to receive Los treatment until they were 8 weeks old. Los was administrated once daily by gavage at the dose of 30mg/kg/d.16-week-old male offspring rats were used to conduct the experiment, with 8 rats in each group.(2) BP,morphology and function of left ventricle, ATRAP gene expression: Systolic BP(SBP) in the conscious offspring rats was measured by the tail-cuff method.Cardiac hemodynamics was evaluated with left cardiac catherization,including indicators ofcardiac contraction function : maximal rate of increase of leftventricle pressure development(LV+dp/dt max), left ventricle end systolic pressure(LVESP)and indicators of cardiac diastolic function:maximal rate of decrease of leftventricle pressure development(LV-dp/dt max),left ventricle end diastolicpressure(LVEDP),time constant of left ventricular isovolume relaxation(T).Body weight(BW) and left ventricle mass(LVM) including ventricular septum weretaken using balance, and LVM/BW was calculated to indicate LVH. After Sirius Red staining, the collagen volume fraction(CVF) in left ventriclesections, an index of cardiac fibrosis, was determined by Image Pro Plus 6.0 software. ELISA was used to evaluate the concentration of Ang II, collogan Igel(Col I) and collagen III gel(Col III) in plasma and myocardium. m RNA and protein expression of ATRAP were detected by real-time RT-PCR and Western blot.(3) DNA methylation profile:With the help of online tool Meth Primer,Cp G island in ATRAP gene promoter was predicted, and primers related to methylation analysis were designed.Thequantitative methylation of nine Cp G sites in the upstream of ATRAP gene transcript start site was assessed by bisulfite sequencingpolymerase chain reaction(BSP). The expression of DNA methyltransferase(DNMT)1, DNMT3 a and DNMT3 b were determined by Western blot. Chromatin immunoprecipitation assay(Chip assay) was used to analyze the binding of DNMTs to ATRAP gene. 2. Cell experiment Firstly, Ang II was used to stimulate H9c2 cells. The rate of protein synthesis, ATRAP expression, methylation level of ATRAP gene promoter, DNMT1,DNMT3 a,DNMT3b expression and the binding of these DNMTs to ATRAP were analyzed by the above mentioned method, respectively; Secondly, H9c2 cells were treated with Los or DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine(5-Aza), Los metabolite(5-carboxylic acid losartan, EXP-3174)prior to stimulation with Ang II, the above indicators were observed again.3Hleucine incorporationwas used to evaluate the protein synthesis. 3. Statistics Data are presented as mean ± SD. Statistical analyses were performed using One-way ANOVA in animal experiment and factorial analysis or One-way ANOVAin cellexperiment, followed by LSD test, and P<0.05 was considered statistically significant. SPSS 19.0 software was used to conduct these analyses.Results 1. At 16 weeks of age, compared with SHRUngroup, in the SHRPre,SHRPost and SHRSeqgroup(1) SBP was lower [(189±20),(174±16),(173±17)vs.(212±22) mm Hg,P<0.05].(2) LVM/BW was significantly reduced[(2.72±0.24),(2.45±0.24),(2.43±0.28)vs.(3.12±0.28)mg/g, P<0.05].(3)LV-dp/dt max was higher、T、LVEDP were lower.(4) CVF, Col I and Col III in myocardium were prominently reduced.(5) Cardiac Ang II level was prominently decreased, and m RNA and protein expression of ATRAP were upregulated(0.10±0.02,0.12±0.02,0.13±0.04 vs. 0.04±0.01,P<0.05). 2. One Cp G island was identified in ATRAP gene promoter. Cardiac methylation levels in four Cp G sites in the upstream of ATRAP gene transcript start site were higher in SHRUngroup than WKYUn group. SHRPre,SHRPost and SHR Seqgroup demonstrated lower methylation levels across the four Cp G sites in ATRAP promotercompared with SHRUn. There were no difference in DNMT1,DNMT3 a,DNMT3b expression in myocardium among five groups of adult rats. Chip assay showed that the bindings of DNMT1 and DNMT3 a to ATRAP gene were higher in SHRUn groupthan WKYUngroup(DNMT1: 2.9±0.3 vs. 1.0±0.0; DNMT3a: 3.0±0.3vs. 1.0±0.0,P<0.05). Offspring rats in SHRPre,SHRPost and SHR Seq demonstrated lower bindings compared with SHRUngroup(DNMT1: 0.9±0.1, 0.9±0.1, 1.0±0.1vs. 2.9±0.3; DNMT3a:1.0±0.2,1.1±0.2,1.1±0.2vs.3.0±0.3,P<0.05). 3. The rate of protein synthesis was significantly increased, and DNMT1 and DNMT3 a expression were upregulated in H9c2 cells in response to Ang II. Chip assayalso showed that higher bindings of DNMT1, DNMT3 a to ATRAP gene in H9c2 cells induced by Ang II, concomitant with the increase in methylation levels across five Cp G sites in ATRAP gene promoter. These effects of Ang II on H9c2 cells were reversed by Los, 5-Aza and EXP-3174 to different extent.Conclusions 1. Early treatment of SHR with Los could persistently postpone the increase in BP, inhibit LVH and fibrosis, ameliorate cardiac dysfunction, lower the level of cardiac Ang II and upregulate ATRAP expression.2. SHR demonstrated higher bindings of DNMT1 and DNMT3 a to ATRAP gene in myocardium, and increased methylation level across multiple Cp G sites in ATRAP gene promoter. Early treatment with Los could partly reverse the changes in myocardium of SHR. 3. Los could inhibite the downregulation of ATRAP expression and increase in the rate of protein synthesisinduced by Ang II,which was associated with the decreasein DNMT1 and DNMT3a-mediated ATRAP methylation. 4. Methylation of ATRAP is associated with the regression of LVH in early losartan treated SHR,and epigenetics is one of possible reason of “missing heritability”.