The Effect Of Lysine-specific Demethylase 1 On Myocardial Regeneration In Neonatal Mice

Background The damaged cardiomyocytes can not regenerate or have only a weak ability to regenerate.Congenital heart regeneration was first found in zebrafish heart,and then Porrello et al found that cardiomyocytes in newborn mice have a strong ability to regenerate.The purpose of the study of congenital myocardial regeneration is to find regulators for cardiomyocyte proliferation,repair and regeneration,so as to improve the prognosis of severe heart disease.Lysine specific demethylase 1(LSD1)is a Flavindependent monoamine oxidase,which can regulate the expression of cell cycle-related genes by removing the methylation of histone H3 monomethylated or dimethylated lysine 4(K4me1/ K4me2)or lysine 9(K9me1/K9me2),and affect the cell cycle progression.It is not clear whether LSD1 is involved in the regulation of congenital heart repair and regeneration.Studies have shown that some Lnc RNA can regulate gene expression in epigenetics by histone modification,including methylation,acetylation and phosphorylation.HOTAIR(HOX transcript antisense RNA)is a protective Lnc RNA,of the heart,and its 3’-terminal domain can bind to LSD1 / Co REST / REST complex to coordinate the regulation of H3K4me1 / H3K4me2 or H3K9me1 / H3K9me2)demethylation.Little is known about the role of HOTAIR in the proliferation,repair and regeneration of mammalian cardiomyocytes.This study focuses on the effect of LSD1 on congenital heart repair and regeneration,and whether HOTAIR participates in the mechanism of LSD1 on myocardial repair and regeneration.Objective 1.To investigate whether LSD1 affects cardiac regeneration in neonatal mice.2.To further study the possible mechanism of the effect of LSD1 on cardiac repair and regeneration in neonatal mice,and to provide a new understanding for exploring the mechanism of cardiomyocyte repair and regeneration.Methods 1.The expression levels of LSD1 and HOTAIR in the myocardium of mice of different ages were detected by q PCR and Western Blotting to verify whether their expression in the heart was related to age.2.To establish the model of apical resection in neonatal mice.Apical resection was performed on P0 mice(within 24 hours after birth).After 21 days,the cardiac function was detected by ultrasound and the size of apical scar tissue was detected by Masson staining to verify the ability of cardiac repair and regeneration in neonatal mice.3.The lentivirus expression vectors of PLKO.1-sh-LSD1 and PLKO.1-sh-HOTAIR were constructed,and the lentivirus was packaged to infect the myocardial tissue of mice to achieve the low expression of LSD1 or HOTAIR in the myocardial tissue.The inhibition results were verified by q PCR and Western Blotting.4.The cardiac function was detected by ultrasound and the size of scar tissue was identified by Masson staining to verify whether knocking down LSD1 or HOTAIR would affect heart repair and regeneration.Furthermore,immunofluorescence assay was used to detect the expression of cell proliferation factors Brdu,Ki67,Ph3 and α-actinin to explore the effect of low level of LSD1 or HOTAIR on cardiomyocyte proliferation.Results 1.The expression levels of LSD1 and HOTAIR in postnatal mouse myocardium were time-dependent.The results of q PCR showed that the m RNA expression levels of LSD1 and HOTAIR in the myocardium of normal neonatal ICR mice were higher on the 1st day,4th day and 7th day,and decreased significantly on the 14 th day.WB results showed that the protein expression level of LSD1 was also at a high level during the first week of birth and decreased at 14 days after birth.2.On the 21 st day after apical resection,the myocardial tissue of neonatal mice could be regenerated completely.The results of Masson staining showed that the cardiac apical tissue basically recovered and there was only a small amount of scar in the adhesion to the chest wall.The results of echocardiography showed that the cardiac function was in the normal range 21 days after apical resection.3.Lentivirus successfully infected mice and down-regulated LSD1 and HOTAIR in myocardial tissue.The sequencing results showed that the target gene sequence of sh oligonucleotide was successfully inserted into PLKO.1 vector.The results of q PCR showed that the m RNA levels of LSD1 and HOTAIR in the myocardium of mice infected with the virus decreased significantly.4.Inhibiting the level of LSD1 or HOTAIR in myocardial tissue can inhibit myocardial regeneration and cardiomyocyte proliferation,but has no significant effect on cardiac function in the short term.The results of Masson staining showed that the damaged myocardium in LSD1 KD group and HOTAIR KD group was replaced by a large number of scar tissue hyperplasia,while there was only a small amount of scar tissue in the apical part of NC group.The results of ultrasound showed that there was no difference in cardiac function between LSD1 KD group and HOTAIR KD group and NC group.The proliferation of cardiac myocytes was detected by immunofluorescence staining of cell proliferation-related factors Brdu,Ki67 and p H3,combined with α-Actinin co-localization analysis.The confocal map of immunofluorescence showed that the number of Ki67,p H3 positive cardiomyocytes in LSD1 KD group and HOTAIR KD group decreased significantly,and the number of Brdu positive cardiomyocytes in NC group was also significantly lower than that in NC group.5.Inhibiting the expression of HOTAIR in cardiomyocytes can decrease the expression level of LSD1 and the demethylation activity of H3K4me2.The results of q PCR and Western Blotting showed that the m RNA and protein expression of LSD1 in myocardial tissue decreased in,HOTAIR KD group.Further study found that down-regulating the level of HOTAIR in myocardial tissue mainly inhibited the demethylation activity of LSD1 to H3K4me2,but did not affect the level of H3K9me2.Conclusions 1.The cardiac injury of newborn mice can activate the strong proliferative potential of cardiomyocytes.The expression level of lysine specific demethylase 1(LSD1)is higher in one week after birth,and then begins to decrease,which is close to the time window of congenital heart regeneration in neonatal mice.2.HOTAIR may regulate the expression of cell cycle genes by affecting the demethylation activity of LSD1 to H3K4me2,thus affecting the division and proliferation of myocardium,resulting in the incomplete regeneration of heart.