Part ? Histone Acetylation Was Involved In Regulating The Low Expression Of Pde4d In CTnIR193H Restrictive Cardiomyopathy Mice

PART ? CTNIR193 H MUTATION RESULTED IN ABNORMAL CALCINEURIN GENE EXPRESSION IN RESTRICTIVE CARDIOMYOPATHY MICEObjective: cardiac troponin I(cTnI)R193H gene mutation can lead to restrictive cardiomyopathy(RCM),and increased calcium sensitivity of myocardial cells is a key factor in active diastolic dysfunction of RCM myocardium,but its mechanism is unknown.Phosphodiesterase(PDE)is an important factor regulating the calcium sensitivity of cardiac myocytes.It can hydrolyze the function of intracellular cAMP,affect PKA and many calmodulin downstream,and thus directly affect the calcium sensitivity of cardiac myocytes.Therefore,this part of the study will clarify the expression of PDE and the expression of Ca2+ regulating molecules or genes such as cAMP and PKA in the myocardium of cTnIR193 H RCM mice.Methods:(1)cTnIR193H + cTnI-knockout(193+KO)mice were generated by hybridization of cTnIR193H+ cTnI-knockout(193+KO)mice and cTnI-knockout C57 mice,193 mice and 193+KO mice of the wild type.The cardiac function and heart rate of mice in each group were detected by high-frequency ultrasound.(2)The expression of genes related to the myocardial adrenergic signaling pathway and the phosphorylation levels of PKA and Ryr2 were detected by q-PCR and western blot.The level of cAMP in myocardial tissue of each group was detected by Ellisa assays.The activation of adrenergic signaling pathway was comprehensively evaluated.(3)Changes of PDE in myocardium of mice in each group were detected by q-PCR and WB,and the relationship between activation of ?-adrenergic signaling pathway and PDE gene expression was determined.(4)Primary myocardial cells were used as the research object,and the free calcium level of myocardial cells with low PDE4 D expression was detected by flow cytometry,as well as the change of the beating rate of myocardial cells with low PDE4 D expression and overexpression of cTnIR193 H.Results:(1)Heart rate of 193 mice and 193+KO mice increased faster than that of wild-type C57 mice.(2)Q-PCR results showed that,compared with the wild-type group,the expressions of adrenergic signaling pathway related genes,such as PKAc?,PKAc? and Ryr2,were significantly increased in the 193+KO group.WB results showed that the phosphorylation levels of PKA and Ryr2 proteins in myocardium of the 193+KO group were significantly higher than those of the control group.The results of Ellisa showed that cAMP level in myocardial tissue of 193+KO group was significantly increased.(3)Q-PCR results showed that PDE gene subtypes PDE1 B,PDE2A,PDE3 A and PDE4 D were all decreased in the myocardial tissue of 193+KO group,and the decrease of PDE4 D was more obvious.WB results suggested that the myocardial PDE4 D protein level in the 193+KO group was decreased.(4)After the expression of PDE4 D was decreased in primary myocardial cells,the intracellular free calcium level was significantly increased.Both primary myocardial cells with overexpression of cTnIR193 H and primary myocardial cells with low expression of PDE4 D showed increased beating.Conclusion:(1)Heart rate of cTnIR193 H mutant RCM mice was faster than that of wild-type mice.(2)The reason for the increased heart rate in cTnIR193 H mutant RCM mice may be related to the activation of adrenergic signaling pathway.(3)Activation of myocardial adrenergic signaling pathway in cTnIR193 H mutant RCM mice may be associated with low expression of PDE gene.(4)Low expression of PDE4 D and cTnIR193 H can increase the pacemaker of myocardial primary cells.Objective: The first part of the study found that the low expression of PDE4 D in cTnIR193 H mutant RCM mice may be the main cause of PKA-related Ca2+ regulating gene changes.However,the mechanism of low expression of PDE4 D is not clear.Histone modification is one of the common regulatory mechanisms of epigenetics,and studies have shown that PDE is regulated by histone modification.In this part,cTnIR193 H mutant RCM mice were taken as the research object to explore the regulatory mechanism of histone modification related to the decreased myocardial PDE4 D in RCM mice.Methods:(1)Similarly,the levels of histone acetylation and methylation in the promoter region of PDE4 D gene in the myocardium of mice in each group were detected by Ch IP assays.(2)m RNA of each group was extracted and reversely transcribed into c DNA,and gene expression levels of various histone acetylases,deacetylases and methylases were detected.(3)The fresh heart tissue proteins of each group were extracted,and the histone modification enzymes that could interact with cTnI were screened by Co IP assays.(4)The binding levels of HDAC1 and SMYD1 in the promoter region of PDE4 D gene in the myocardium of mice in each group were detected by Ch IP assays.Results:(1)Compared with the control group,the acetylation levels of H3K4 and H3K9 in the PDE4 D promoter region of 193+ko group and the level of H3K4 trimethylation were significantly reduced,and the differences were statistically significant.(2)Histone deacetylase HDAC8 and HDAC9 genes were increased and HDAC3 expression was decreased in 193 mice compared with the control group.Histone acetylase GCN5 and histone acylase SMYD1 were increased in 193 mice,and the difference was statistically significant.(3)In myocardial tissue,cTnI may bind to HDAC1 and SMYD1.(4)Compared with the control group,the binding water of HDAC1 and SMYD1 in the promoter region of PDE4 D gene in the myocardium of 193 mice was increasedConclusion:(1)cTnI can interact with HDAC1 and SMYD1 in various histone modification enzymes.(2)The reduction of PDE4 D gene in the myocardium of cTnIR193 H mutant RCM mice may be related to the reduction of acetylation levels of H3K4 and H3K9 in the promoter region and the trimethylation level of H3K4.(3)Changes in acetylation levels of H3K4 and H3K9 in the PDE4 D promoter region may be associated with HDAC1.PART ? CTNIR193 H REGULATES THE LOW EXPRESSION OF PDE4 D THROUGH HDAC1-MEDIATED HISTONE ACETYLATION MODIFICATIONObjective: Part ? suggests that the low expression of PDE4 D may be related to hdac1-mediated histone acetylation.As a key gene limiting the pathogenesis of cardiomyopathy,cTnI can interact with HDAC1.Therefore,whether the cTnIR193 H mutation may regulate the low expression of PDE4 D through HDAC1 mediation? Based on further verification of HDAC1's regulation of PDE4 D,this part will clarify the internal mechanism of cTnIR193H's regulation of PDE4 D.Methods:(1)The expression of PDE4 D gene and protein was detected in primary myocardial cells after overexpression of HDAC1.(2)After overexpression of HDAC1,the binding level of HDAC1 and the acetylation level of H3K4 and H3K9 in the promoter region of PDE4 D gene were detected.(3)cTnI was confirmed to be nuclear by immunofluorescence.After overexpression of wild-type cTnI and cTnIR193 H,WB was used to observe the ratio.(4)The transcriptional activity of cTnI and cTnIR193 H on PDE4 D promoter region was verified by luciferase reporter gene assay.(5)After overexpression of cTnI and cTnIR193 H,the expression level of PDE4 D protein was observed.(6) After overexpression of cTnI and cTnIR193 H,the binding levels of cTnI,cTnIR193 H and HDAC1 in the promoter region of PDE4 D gene were observed by Ch IP assay.(7)After overexpression of cTnI and cTnIR193 H,the binding ability of cTnI and HDAC1 was detected by Co IP technology.Results:(1)Compared with the control group,the level of PDE4 D protein in the HDAC1 overexpression group was significantly decreased.(2)Compared with the control group,the HDAC1 binding level in the promoter region of PDE4 D gene in the HDAC1 overexpression group was significantly increased,and the acetylation levels of H3K4 and H3K9 were significantly reduced,with statistically significant differences.(3)cTnI can be observed in the myocardial nucleus,and WB results suggest that the amount of cTnI and cTnIR193 H in the cell nucleus accounts for about 40% of the total cTnI or cTnIR193 H.(4)Compared with the control,the fluorescence value of cTnI on the upstream of PDE4 D promoter was increased.However,cTnIR193 H did not significantly change the fluorescence value of 2000 b upstream of PDE4 D promoter.(5)Compared with the control,the expression of PDE4 D gene and protein in cTnIR193 H group was significantly reduced.Compared with control,the m RNA level of PDE4 D in the cTnI group was significantly increased,but the protein level was not significantly changed.(6)Compared with the cTnI group and the cTnIR193 H group,both of them could bind to the PDE4 D promoter region.Compared with the cTnI group,the binding level of HDAC1 in the PDE4D promoter region in the cTnIR193 H group was significantly increased,and the difference was statistically significant.(7)Co IP results suggested that,compared with cTnI,the amount of cTnIR193 H combined with HDAC1 increased significantly,and the difference was statistically significant.Conclusion:(1)HDAC1 inhibits PDE4 D expression by regulating histone H3K4 and H3K9 acetylation levels.(2)The ability of cTnI and cTnIR193 H to enter the nucleus was the same.(3)cTnI has transcriptional activity to PDE4 D.(4)cTnIR193 H can reduce the expression of PDE4 D gene and protein.cTnI can promote the expression of PDE4 D gene.(5)Both cTnI and cTnIR193 H can bind to the promoter region of PDE4 D with the same binding capacity,but cTnIR193 H is more likely to bind HDAC1,thus increasing the binding level of HDAC1 in the promoter region of PDE4 D.PART ? EGCG IMPROVES THE LOW EXPRESSION OF PDE4 D INDUCED BY CTNIR193 H MUTATION BY INHIBITING THE BINDING OF CTNIR193 H TO HDAC1Objective: Part ? suggests that HDAC1 mediates the regulation of PDE4 D low expression by cTnIR193 H.Our previous study found that EGCG can inhibit hdac1-mediated histone acetylation modification to improve gene expression,and EGCG significantly improved the cardiac function of cTnIR193 H restricted cardiomyopathy mice.Therefore,EGCG was selected as HDAC1 inhibitor in this part to further verify the histone modification regulation mechanism of hdac1-mediated cTnIR193 H down-regulation of PDE4 D expression.Methods:(1)The primary myocardial cells were divided into idled group,cTnIR193 H group and cTnIR193H+EGCG intervention group,and the expression of PDE4 D gene and protein were detected by q-PCR and WB.(2)HDAC1 binding level and cTnIR193 H binding level in PDE4 D promoter region of each group were detected by Ch IP.(3)The effect of EGCG on the binding force of cTnIR193 H and HDAC1 was tested by Co IP.(4)The effect of EGCG on HDAC1 activity was detected by Co IP.Results:(1)Compared with the control group,PDE4 D gene and protein levels were decreased in the cTnIR193 H group,and the expression level of PDE4 D was significantly increased after EGCG intervention compared with the cTnIR193 H group.(2)Compared with the control group,the binding amount of HDAC1 in the PDE4 D promoter region of cTnIR193 H group increased.Compared with the cTnIR193 H group,the binding levels of cTnIR193 H and HDAC1 in the PDE4 D promoter region of the cTnIR193 H group were significantly reduced after the intervention of EGCG.(3)Co IP results showed that compared with the cTnIR193 H group,the binding capacity of cTnIR193 H and HDAC1 was significantly reduced after EGCG intervention,and the difference was statistically significant.(4)There was no significant change in the activity of HDAC1 in each group.Conclusion:(1)EGCG can alleviate the low expression of PDE4 D caused by cTnIR193 H.(2)EGCG reduces the binding ability of cTnIR193 H to HDAC1.(3)EGCG inhibits the binding level of cTnIR193 H and HDAC1 in the PDE4 D promoter region.(4)EGCG had no effect on HDAC1 level and activity of HDAC1 in cTnIR193H-overexpressed myocardium.