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ESTABLISHMENT OF MINIMALLY INVASIVE MOUSE TRANSVERSE AORTIC CONSTRICTION HEART FAILURE MODEL WITHOUT MECHANICAL VENTILATION Objective:The transverse aortic constriction(TAC)heart model is widely used in the research of pressure overload induced heart failure because of its advantages of short establishment time and good repeatability.In the past,tracheal intubation and mechanical ventilation were mostly used to establish this animal model.Endotracheal intubation was time consuming in mice,repeated attempts can result in airway damage,and the classic TAC operation was highly traumatic,resulting in a high mortality in mice.To reduce the airway injury and surgical trauma induced death in mice,theTAC model was established without endotracheal intubation and with minimally invasive surgery.Methods:1.c57 mice aged 3 months were divided into sham operation group,mechanical ventilation TAC group and non-mechanical ventilation TAC group to establish the TAC model.In the mechanical ventilation TAC group,the classic TAC surgery was performed under mechanical ventilation with endotracheal intubation.In the non-mechanical ventilation TAC group,minimally invasive TAC surgery was performed with spontaneous breathing;In the sham group,the surgical procedure was the same as that of the non-mechanical ventilation TAC group except contraction of aortic arch;2.In the sham group,mechanical ventilation TAC group and non-mechanical ventilation TAC group,high frequency ultrasound was used to detect aortic arch diameter,blood flow velocity and pressure gradient before and 3 days after surgery.Before and 4 weeks after operation,LVAWd,LVIDd and LVPWd were detected respectively,and the LVEF was calculated.The left ventricular myocardial tissue of mice were collected 4 weeks after surgery,and the ultrastructure of myocardium was observed by microscope after HE staining;3.In the sham group and non-mechanical ventilation TAC group,LVAWd,LVIDd and LVPWd were detected respectively,and the LVEFwas calculated before and 8 weeks after operation;LW and BW were measured 8 weeks after surgery,and LW/BW values were calculated.Results:1.The operating time including endotracheal intubation time in non-mechanical ventilation TAC group was 10.86±2.36 minutes,which was significantly reduced compared with that in mechanical ventilation TAC group(24.44±3.55 minutes)(p<0.05),the survival rate of mice were improved,93.3% and 73.3% respectively(p<0.05);2.There was no significant difference in arterial arch diameter,blood flow velocity and pressure gradient,LVAWd,LVIDd,LVPWd and LVEF among sham group,mechanical ventilation TAC group and non-mechanical ventilation TAC group before operation.Postoperatively,there were no statistically significant differences between mechanical ventilation TAC group and the non-mechanical ventilation TAC group in the above indicators at 3 days or 4 weeks after surgery(p>0.05),but the difference was statistically significant compared with themselves preoperatively and sham group postoperatively(p<0.05).Compared with the sham group,cardiac myocytes in the mechanical ventilation TAC group and the non-mechanical ventilation TAC group were enlarged(p<0.05);3.Eight weeks after surgery,LVIDd 3.89±0.04 mm,LVEF45.07±4.19% in non-mechanical ventilation TAC group were statistically different compared with itself preoperatively(LVIDd 3.69±0.05 mm,LVEF 62.35±2.20%)and in sham group postoperatively(LVIDd3.71±0.04 mm,LVEF 61.93±2.28%)(p<0.05).The LW/BW of TAC without mechanical ventilation group was 8.15+0.58 at 8 weeks after operation,which was 55.8% higher than that of sham group(5.23+0.22).Conclusions:1.Minimally invasive TAC without mechanical ventilation in mice can reduce operation time and improve survival rate;2.Eight weeks after operation,the TAC heart failure model can be established successfully by minimally invasive surgery without mechanical ventilation.PART ? HISTONE ACETYLATION MECHANISM OF SERCA2 A LOW EXPRESSION IN HEART FAILURE MICEObjective:In the first part,we successfully established a minimally invasive Transverse Aortic Constriction(TAC)heart failure model of mice.Sarcoplasmic reticulum Ca2+-ATPase 2a(SERCA2a)is an important functional protein of the heart.The expression of SERCA2 a protein and its m RNA in heart failure is significantly reduced,suggesting that the decrease of SERCA2 a may occur at its transcriptional level.Histone modification can regulate gene transcription and expression,previous studies found that histone modification can regulate the expression of ATP2a2 which encoding SERCA2 a,increased HDAC1 expression in the heart reduces histone acetylation levels.Therefore,we hypothesized that the low expression of SERCA2 a in heart failure might be related to the decrease of HDAC1-mediated histone acetylation modification,we design this part to investigate histone acetylation mechanisms of SERCA2 a expression.Methods:The mouse heart tissue obtained 8 weeks after surgery in the first part was tested as follows:1.The expression levels of SERCA2 a m RNA and HDAC1 m RNA were detected by Q-PCR;2.ChIP-Q-PCR was used to detect level of histones Ac H3,Ac H3K4 and Ac H3K9,the binding levels of HDAC1,GATA4 and Mef2 c near the proximal promoter region of Atp2a2.Results:1.The expression level of myocardial SERCA2 a m RNA in TAC group was significantly lower than that in the sham group(p<0.05),the expression level of myocardial HDAC1 m RNA was significantly higher than that in the sham group(p<0.05);2.The levels of Ac H3 and Ac H3K9 near the proximal promoter region of Atp2a2 in TAC group were significantly lower than those in the sham group(p<0.05).The binding level of HDAC1 near the proximal promoter region of Atp2a2 was significantly higher in the TAC group than in the sham group(p<0.05).The binding level of GATA4 and Mef2 c near the proximal promoter region of Atp2a2 was significantly lower in the TAC group than in the sham group(p<0.05).Conclusions:The decrease of histone H3K9 acetylation near the proximal promoter region of Atp2a2 in heart failure mice may be related to the increase of HDAC1 m RNA expression level,which may result in the decrease of binding of transcription factors GATA4 and Mef2 c near the proximal promoter region of Atp2a2 and decrease the expression of SERCA2 a,suggesting that the acetylation of histone H3K9 near the proximal promoter region of Atp2a2 may be involved in the regulation of SERCA2 a expression.PART ? EGCG INHIBITED HDAC1,UPREGULATED SERCA2 A EXPRESSION AND PREVENTED HEART FAILURE IN TAC MICEObjective:In the second part,we found that the expression of SERCA2 a was significantly decreased in mice with heart failure.Increased expression of HDAC1 leads to decreased acetylation of histone H3K9 near the proximal promoter region of Atp2a2,which may play an important role.Our previous study found that EGCG can inhibit HDAC1 up-regulate the acetylation level of histone H3K9.Therefore,EGCG was selected as the intervention agent in this part to clarify the effect of EGCG on SERCA2 a expression and cardiac function in TAC mice.Methods:c57 mice aged 3 months were randomly divided into sham operation group(SHAM group),non-mechanical ventilation TAC group(TAC group),TAC+EGCG group and SHAM+EGCG group.The TAC+EGCG group and SHAM+EGCG group were given intraperitoneal injection of EGCG 50mg/kg/d for 12 weeks.SHAM group and TAC group were injected with normal saline for control.Ultrasound was used to evaluate the cardiac function of mice.Western blot and Q-PCR were used to detect SERCA2a protein and m RNA expression levels in the hearts of mice.The activity of HDAC1 in myocardial tissue of each group was detected.The acetylation level of histone H3?H3K4?H3K9,the binding level HDAC1,GATA4 and Mef2 c near the proximal promoter region of Atp2a2 were detected by ChIP-Q-PCR.Results:1.Twelve weeks after surgery,LVIDd 3.80±0.13 mm,LVEF62.24±3.07% in TAC+EGCG group had no significant changes compared with itself preoperatively(LVIDd 3.73±0.09 mm,LVEF 63.01±1.79%)(p>0.05).In TAC group,LVIDd 4.04±0.05 mm was increased compared with that before operation(3.73±0.09mm),LVEF 41.37±7.79% was decreased compared with that before operation(62.99±1.81%)(p<0.05).The LW/BW of TAC group was 8.51±0.25 at 12 weeks after operation,which was 64.9% higher than that of sham group(5.16±0.10);The LW/BW of TAC+EGCG group was 5.29±0.09,the difference was not statistically significant compared with the sham group(p>0.05);2.SERCA2 a protein and m RNA levels in the TAC+EGCG group was increased compared with the TAC group(p<0.05),but there was no significant change compared with that in the SHAM group(p>0.05);3.The level of Ac H3 and Ac H3K9,binding level of GATA4 and Mef2 c near the proximal promoter region of Atp2a2 in TAC+EGCG group were increased compared with TAC group,but there was no significant difference between the TAC+EGCG group and SHAM group(p > 0.05);4.The activity of HDAC1 and the binding level of HDAC1 near the proximal promoter region of Atp2a2 in the TAC+EGCG group were lower than that in the TAC group(p<0.05),but there was no statistical difference between the TAC+EGCG group and SHAM group(p>0.05).Conclusions:1.EGCG can prevent heart failure in TAC mice;2.EGCG may inhibit HDAC1 activity in the heart of TAC mice,thereby reducing its binding level near the proximal promoter region of Atp2a2,causing increased acetylation of histone H3K9 near the proximal promoter region of Atp2a2,promoting the binding of the corresponding transcription factors GATA4 and Mef2 c,and preventing the low expression of SERCA2 a. |
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