| BackgroundFollowing with the scientific-technique progress,the faster pace of life is and the change of dietary structure,the morbidity of cardiovascular diseases is increasing years by years,more over it shows a younger tendency.As a common disease,cardiac hypertrophy is an inherited disease.It can be caused by various factors,especially long time arterial hypertension,which is known as pressure overload cardiac hypertrophy.it is related to heart failure,then result in a sudden death.Cardiac hypertrophy is a clinic symptom of variety diseases.Recent years,effect of abnormal epigenetic mechanisms in the pathogenesis and development of various disease has been paid general attention.Methylation which is an important measure of regulating genomic function is a mainly form of epigenetic modification of genome deoxyribonucleicacid.Lysine specific demethylase 1(LSD1)is a specific demethylase that was first discovered.LSD1 can specificly catalyze demethylation on monomethylated and dimethylated histone of the fourth and the ninth lysine on H3(H3K4 / H3K9).LSD1 is reported to have relationship with different kinds of tumour.It seems that the epigenetic changes of LSD1 may play very important roles in the development of many kinds of tumour.However,the role of LSD1 on the process of developing cardiac hypertrophy has not yet been elucidated.This study make pressure-overload cardiac hypertrophic rat model by the transverse ascending aorta constriction operation,in order to investigate the relationship between LSD1 and pressure-overload cardiac hypertrophy,lay the foundation of displaying the mechanism and further treatment of cardiac hypertrophy.Objective1.To explore the role of LSD1 on the occurrence of pressure-overload cardiac hypertrophy,and understand the effect of histone methylation state on pressure-overload cardiac hypertrophy,thereby provide theoretical basis for regarding LSD1 as a new target of clinical treatment of cardiac hypertrophy.2.To construct and evaluate the level of rat-specific LSD1 overexpression plasmid and demethylation disfunction plasmid in HEK293 T cells.Meanwhile toprovide a new method for the further review of the role of histone methylated modification on cardiac hypertrophy.Methods1.Healthy male SD rats(120 ~ 130 g)were feeding as SPF experiment animals.They were randomly seperate to three groups: the Normal control group(Control),the Sham operation group(Sham)and the transverse aortic constrictiongroup(TAC).Each group contain three rats.We measured the cardiac hypertrophy phenotype?hypertrophy factors?LSD1 and the downstream proteins by variety experiment methods include heart weight ratio?Echocardiography?Western Blot?qPCR?H&E staining and Immunohistochemistry.2.Through PCR amplified LSD1 and overlap PCR amplified demethylation disfunction fragment to construct the rat-specific LSD1 overexpression plasmid and LSD1 demethylation disfunction plasmid.To verify the plasmids by agarose-gel-electrophoresis and nucleotide sequencing methods.Packaged the two plasmids which had been verified and the blank vector to the virus.Use the virus to infect corresponding HEK293 T cells.Add puromycin to sieve stably expressive cells.Demonstrate the LSD1 expressive level in HEK293 T cells by Western Blot and RT-qPCR.Results1.The changes of LSD1 expression in heart tissues of pressure-overload cardiac hypertrophy ratsHeart rates and Blood Pressures results showed that: compared with the Control and Sham group,both the heart rates and blood pressures of the rats from TAC group were significantly higher.the heart rates and blood pressures of the sham group rats showed a reduction when compared with the control group rats.(n=6,*p < 0.05?***p< 0.0001).Echocardiography found an obvious cardiac hypertrophy in rats of TAC group.The leftventricular posterior wall and left ventricular mass of rats from TAC group were significantly thick compared with those of the rats from Control group and Sham group.The ventricular cavity was decreased,while the ejection fraction andfraction shortening were increased in rats of TAC group(n=6,***P<0.001 ?**P<0.01).The results of the cardiac phenotype showed that the hearts of rats from the TAC group was significantly larger than those rats from Control group and Sham group.The heart weight ratio of TAC group rats obviously increased.The results were statistically significant(n=6,***p < 0.0001).H & E staining showed that the cardiac cells of rats from TAC group were significantly hypertrophied compared with the Control group and Sham group in cell cross sectional area..The expression of three types of hypertrophic factors: The qPCR results showed that compared with the Control group and Sham group,the mRNA expression of three cardiac hypertrophic factors(Myosin Light Chain 2v,MLC-2V;Alpha myosin Heavy chain,?-MHC;Atrial Natriuretic Peptide,ANP)of the heart tissue of TAC group rats were significantly up-regulated.(n=6,**p < 0.01?***p < 0.001)The expression of LSD1 and the corrected histones downstream in heart tissues:the results of qPCR?Western Blot and Immunohistochemistry showed that compared with Control group and Sham group,both the mRNA and the protein expression level of LSD1 in the heart tissues of TAC group rats were significantly decreased(n=6,**p < 0.01),while the expression of downstream histones(H3K4me1?H3K4me2?H3K9me1 and H3K9me2)were significantly increased.(n=6,**p < 0.01?***p <0.001 p < 0.01).2.The result of construction rat-specific LSD1 overexpression plasmid and demethylation disfunction plasmid: Western Blot and qPCR assays showed that plasmids overexpression in HEK293 T cells successfully.The two kinds of plasmids were established to the further study of LSD1 gene and the function of LSD1 demethylation.The results showed that the LSD1 was overexpression from LSD1 overexpression group and demethylation disfunction group compared with control group.The results were statistically significant(**p < 0.01?***p < 0.001).Conclusions:1.LSD1 may closely relate with the development of pressure-overload cardiachypertrophy.Sustained high pressure load may induce pathological cardiac hypertrophy through increasing methylated state of histone by inhibiting the expression of LSD1.2.Successfully constructed specific LSD1 overexpression plasmid and demethylation disfunction plasmid,and achieved overexpression in HEK293 T cells.It established foundation for further study on the relationship between the demethylation function of LSD1 and the various diseases. |
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