The Expression Of Histone Acetyltransferase Subtypes During Mouse Heart Development

Background Congenital heart disease(CHD)is the most common cardiovascular malformation in children and the most frequent birth defects. With the development of imageology and surgery and intervention , there have been great progression in diagnosis and the treatment of CHD, however, the pathogenesis is not clear. At present, the researches about the pathogenesis concentrate on cardiac-related genes. Heart development is a complex process that require the precisely spatiotemporal expression of a series of cardiac-associated genes and the regulation of transcriptional factors[1]. The process is not only decided by the gene sequences, to a greater extent, decided by the alteration of chromatin conformation[2] , which is also called chromatin remodeling that belongs to the category of epigenetic research[3]. However, scant information is available about the epigenetic mechanism in heart development. Therefore, it has greatly scientific significance to clarify the mechanism of cardiogenesis from epigenetic histone acetylation perspective in the level of gene regulation. Epigenetics include DNA modification, histone modification and non-coding RNA regulation[4]. In resent years, studies find that histone acetylation modification plays an important regulation role in embryonic development. Histone acetyltransferases (HATs ) catalyze histone to acetylate which decide the acetylation level of cardiac tissue and affect the timed regulation of cardiac-relaed genes. Therefore, to clarify the epigenetic regulation patterns of heart development, it require to study the spatiotemporal expression of HATs in developmental heart, firstly.Objectives To establish the time-dose curves of HATs subtypes during cardiogenesis by detecting the spatiotemporal expressions of cardiac-related five HATs—p300,CBP,GCN5,PCAF and SRC1 in developmental heart, and explore the relationship between different HATs and heart development.Material and Methods The normal mouse hearts from E7.5~E18 and neonatal 1 day as well as adult were used to intimately describe the morphological changes by anatomic observation and paraffin section HE staining technique. Protein distributions and timed mRNA expressions of p300,CBP and GCN5 were dynamicly detected by immunohistochemistry and RT-PCR technique in heart development; The spatiotemporal expressions of PCAF and SRC1 proteins were dynamicly analyzed by immunohistochemistry and western blot during cardiogenesis, and the time-dose curves of different HATs subtypes were established. Results1. The heart was initiated in crescent-shaped cardiac anlage at about mouse embryo day 7.5 (E7.5). Soon after that, the cardiac crescent converged to form the primary linear cardiac tube at about E8.5 and looping cardiac tube at E9.5. At E10.5, atrioventricular canal formed as well as atrioventricular endocardial cushions (EC) and protrusive trabeculae had been visible. Interatria septum formed at E11.5～E13.5 and interventricular septum formed at E11.5～E14.5 and atrioventricular valve formed at E12.5～E15.5 and outflow tract was septated into pulmonary artery and aorta at E11.5~E13.5. Cardiomyocyte differentiation and multiplication made the walls compact and thicken at E11.5～E16.5 while cardiomyocyte multiplication and increase caused walls to become thicker and the heart to become greater at E16.5～postnatal day 1. During postnatal stage, with the growth of the mouse, heart became big yet cardiomyocyte no multiplication longer. Cardiomyocyte displayed polygon in embryo stage and became rod-shape adult type in postnatal 1 week.2. The spatiotemporal expression of p300 and CBP during heart development: (1)Immunohistochemical results revealed: Strong or relatively strong expressions of p300 were detected in nearly every region of the hearts in the whole developmental stages. No expressions of CBP were detected at E7.5. CBP relatively strongly expressed at cardiac tube at E8.5 and E9.5. At E10.5~E18 and postnatal day 1, CBP was found relatively strong expression in myocardium and weak expression in atrioventricular valves as well as trabeculae and relatively weak expression in muscular interventricular septum and weaker expression in membranous interventricular septum. CBP relatively weakly expressed in myocardium and trabeculae and extremely weakly expressed in interventricular septum at the 3 months mouse. Compared to same stages and same regions of p300, CBP relatively low rate of positive cells expressed, the difference was statistically significant(P<0.05).( 2)RT-PCR results revealed: In the developmental hearts after E10.5, p300 mRNA peakingly expressed E10.5~E11.5; CBP mRNA peakingly expressed ranging from E10.5 to E12.5. They had gradually decreasing expressions in the subsequent heart development time points.3. The spatiotemporal expression of GCN5 during heart development: (1)Immunohistochemical results revealed: No expressions were detected in heart anlages at E7.5~E9.5; GCN5 was found widespread and weak expressions as well as relatively strong expressions in EC at E10.5~E11.5; GCN5 was showed weak expressions in myocardium and relatively strong expressions in EC and membranous interventricular septum and atrioventricular valves at the subsequent development stages. (2) RT-PCR results revealed: In the developmental hearts after E10.5, GCN5 mRNA peaking expression at E10.5~E11.5 and decreasing expression at E12.5~E15.5 and extremely low expression after E16.5 were demonstrated.4. The spatiotemporal expression of PCAF during heart development: (1)Immunohistochemical results revealed: Widespread and weak expressions of PCAF in heart anlages were detected at E7.5~E9.5; PCAF was found relatively strong expression in myocardium, relatively weak expression in endocardium and atrioventricular valves as well as trabeculae, weak expression in muscular interventricular septum and extremely weak expression in membranous interventricular septum. At 3 months adult mouse, PCAF weakly expressed in walls and trabeculae and extremely weakly expressed in septum. (2) Western blot results revealed: In the developmental hearts after E10.5, PCAF proteins relatively highly expressed at E10.5 and reached the peaking expression at E11.5 and gradually decreasingly expressed in the subsequent developmental stages. There are two expression platforms at E13.5~E15.5 and E16.5~postnatal day 1, respectively. At 3 months adult mouse heart, the lowest expression of PCAF was detected.5. The spatiotemporal expression of SRC1 during heart development: (1)Immunohistochemical results revealed: No expression of SRC1 was detected in heart anlage at E7.5; SRC1 was found very weak expression in cardiac tube at E8.5~E9.5; SRC1 was found weak expression in trabeculae and relatively strong and widespread expression in other heart regions after E10.5. (2)Western blot results revealed: In the developmental hearts after E10.5, SRC1 proteins relatively lowly expressed at E10.5 and reached the expression peak at E11.5~E12.5 and gradually decreasingly expressed in the subsequent developmental stages.Conclusion There were different distributions and dynamic expression variants of p300,CBP,GCN5,PCAF and SRC1, indicating that the HATs subtypes participated in cardiogenesis, and different HATs subtypes may be closely related to heart specific structure development.