Epigenetic Mechanisms In CTnI Decrease Caused Cardiac Diastolic Dysfunction

Part ⅠDetection of time-course of cTnI from birth to aging and evaluation of the morphology and function of aging hearts ObjectiveCardiac tropnoin I(cTnI), a development regulated gene, plays a critical role in the regulation of diastolic function, and its low expression may result in cardiac diastolic dysfunction, which is the most common form of cardiovascular disorders in older adults, and is increasing in prevalence as the population ages. In this part, the structure and function of aging hearts were evaluated, and time-course of cTnI expression from birth to aging was detected. MethodHealthy SPF class c57BL6 mice were used as research objects. cTnI expression levels were determined at various ages(new born, 2-week, 3, 10 and 18 months) by Western blot and Q-PCR. Cardiac functions of 3m and 18 m mice were evaluated by high frequency ultrasound. Transmission electron microscopy and H&E staining were used for morphology comparison between 3m and 18 m mice. Result1. Time-course of cTnI protein and m RNA levels increased gradually after birth, and reached peak at 3m, kept stable at 10 m, and decreased significantly at 18m(NB, 2w, 18 m vs. 3m p<0.05; 10 m vs. 3m p>0.05); Protein levels of c Tn T showed no significant changes among each group(p>0.05).2. EF value and FS value showed no significant changes between 3m and 18 m groups’ mice(p<0.05); IVRT of 18 m mice showed a significant prolongation of IVRT and a reduction of E/A value compared with 3m mice(p<0.05).3. Compared with 3m, cardiac ultra structure of mice in 18-month showed an enlargement of sarcoplasmic reticulum, thickening of Z line, sarcomere dissolution and hydropic degeneration; H&E staining showed no significant changes between 3m and 18 m hearts. Conclusion1. cTnI increased gradually after birth, and reached peak level at young adult age(3m), then its level decreased significantly in 18 m both in m RNA and protein levels2. Cardiac diastolic functions of aging mice were reduced compared to young adult mice(3m). 3. TEM showed a destruction of myofilament in aged mice.Part Ⅱ Epignetic mechanisms of cTnI decrese in aging hearts ObjectivecTnI showed a significant decrease both in m RNA and protein level, indicating that low expression of cTnI may occur in its transcription level. DNA methylation regulates gene transcription through methylating on Cp G island or CG sites. Histone acetylation modified chromatin structure dynamically and control gene activation or repression. So, we design this part of experiment to investigate epigenetic modification mechanisms of cTnI expression. MethodHeart tissues of each group were obtained as previous description. BSP and MSP were used to detect the methylation levels of Cp G island in the upstream promoter of cTnI and CG sites of GATA & Mef2 elements. Ch IP assays were employed to demonstrate the acetylated H3, H3K9 and H3K27 levels in the key-cis elements of cTnI promoter. Binding levels of GATA4 and Mef2 c with those elements were detected by Ch IP. Results1. Neither DNA methylation level of Cp G island nor CG sites in the GATA & Mef2 element of cTnI gene’s proximal promoter showed no significant changes among each group(p>0.05);2. Ac H3 levels at 3m were higher than that at newborn and aged stage(p<0.05). Levels of Ac H3K9 and Ac H3K27 reached peak at 3m, however, only Ac H3K9 reduced at 18m(p<0.05).3. Binding levels of GATA4 and Mef2 c increased gradually after birth, and reached peak at 3m, kept stable at 10 m, and decreased significantly at 18m(NB, 2w, 18 m vs. 3m p<0.05; 10 m vs. 3m p>0.05). Conclusion1. DNA methylation may not regulate cTnI expression.2. Histone acetylation modification involved in cTnI decrease in aging hearts.Part Ⅲ EGCG up-regulated cTnI expression and improve heartdiastolic function in aging mice ObjectivecTnI reduced in aging hearts, and histone actylation, especially H3K9 acetylaion may played significant role in cTnI decrease. Recently studies showed that EGCG may increase Ac H3K9. So in this part aged mice were treated with EGCG to investigated whether it could up-regulate cTnI expression and improve heart diastolic function in aging mice. Method16m mice were randomly divided into Bland group(treated with normal saline), DMSO treated group and EGCG treated group. Mice were given a dose of 50mg/kg/d by intraperitoneal injection for 8 weeks. Heart functions of 18m+EGCG group mice were evaluated by high frequency ultrasound. Heart tissue samples of 18 m, 18m+DMSO, 18 m +EGCG and 3m were collected as previous description. WB and Q-PCR were used to detect the expression level of cTnI; HDAC1, HDAC2 and HDAC3 m RNA levels were measured by Q-PCR; Ch IP assays were employed to test the histone acetylation levels of H3K9 in the key-cis elements region of cTnI. Binding levels of GATA4 and Mef2 c with the elements were detected by Ch IP, and binding levels of HDAC1 with the elements were tested also by Ch IP.Results1. The IVRT of 18m+EGCG group were reduced compared with 18 m group(p<0.05), showed no significant differences with 3m group(p>0.05)2. Both cTnI m RNA and protein levels of 18m+EGCG were significant higher than that of 18 m and 18m+DMSO groups(p<0.05), and showed no significant differences compared to 3m;3. EGCG inhibited HDAC1 and HDAC3 m RNA levels.4. Ac H3K9 levels in the key-cis elements of cTnI in 18m+EGCG group were increased compared with 18 m and 18m+DMSO groups, and no significant differences were found when compared to 3m.5. Binding levels of HDAC1 with those elements were raised after EGCG treatment in 18 m, but HDAC2 and HDAC3.6. Binding levels of GATA4 and Mef2 c with the key-cis elements went up after EGCG treatmen. Conclusions1. EGCG treatment might inhibit HDAC1 expression and its binding level with cTnI’s promoter, and then increased cTnI expression levels both in m RNA and protein levels of 18 m mice through increasing the Ac H3K9 levels of cTnI promoter regions. 2. EGCG could improve heart functions of aging mice.