Experimental Study On The Effect And Mechanism Of Elevated Glucose On Heart Development In Zebrafish Embryos

Part 1:The effects of elevated glucose on cardiac development in zebrafish embryosObjective:Congenital heart defects (CHD) are among the most common malformations in fetuses. It is generally understood that both genetic basis and environmental factors are contributed to the highly complex etiology of congenital heart defects. Most of CHD are due to the interplay of genetic abnormalities with environmental factors. A solid body of epidemiologic literature indicates that pregestational diabetes mellitus (PGDM) is a highly significant risk factor for congenital heart defects and hyperglycemia acts as a major teratogen in diabetic pregnancy. However, the direct effects of hyperglycemia on embryonic heart development have not been elucidated. In this study, we have investigated the direct effects of elevated glucose on cardiac development using zebrafish as a model system. Methods:To determine the D-glucose-sensitive window during early heart development, embryos in different stages (6,24 and 36 hpf) were exposed to 25mmol/L D-glucose for 12 or 24 h. The percentage of embryos with cardiac malformations was recorded at 72 hpf. In the subsequent studies, shield stage (6 hpf) embryos were randomly assigned to culture medium that contained either 25 mmol/L D-glucose or L-glucose for 24 h. To quantify the effect of glucose on embryo development rate, the somite number and the head-trunk angle (HTA) were recorded. To evaluate the heart development rate, heart morphogenesis was analyzed by using a specific marker of heart, cardiac myosin light chain 2.Results:The highest incidence of cardiac malformations occurred during 6-30 hpf exposure periods and the malformation incidence was gradually decreased as the exposure initiation time was postponed. Elevated glucose caused severe growth retardation and developmental delay in zebrafish embryos. The heart development is also delayed and approximately synchronized with the whole embryo. Embryos exposed to D-glucose exhibited diverse gross cardiac malformations, including pericardial edema, an unlooped heart and blood regurgitation. In addition, a small fraction of embryos displayed blood toggling between the atrium and ventricle. However, embryos exposed to L-glucose showed no apparent phenotype.Conclusions:The sensitive window for glucose-induced cardiac defects in zebrafish embryos occurs during 6-30 hpf. Elevated glucose alone caused severe developmental delay and diverse cardiac defects in zebrafish embryos.Part 2:The mechanism of cardiac developmental delay caused by elevated glucoseObjective:To elucidate the mechanism of cardiac developmental delay caused by elevated glucose. Methods:The expression of IGFBP-la, IGFBP-1b and IGFBP-2 was measured using in situ hybridization and qRT-PCR. Results:Elevated glucose increased IGFBP-la expression compared with stage-matched controls but has no effect on expression of IGFBP-lb and IGFBP-2. Conclusions:IGFBP-la is involved in the cardiac developmental delay in response to elevated glucose.Part 3:The mechanism of cardiac defects caused by elevated glucoseObjective:To ascertain the origin of the cardiac defects and reveal the underlying molecular mechanisms for cardiac defects caused by elevated glucose.Methods:The early heart morphogenesis was examined using two marker genes, nkx2.5 and cmlc2. The expression of tbx5, tbx20, has2, notch 1b and bmp4 was measured using in situ hybridization.Results:The bilateral heart primordial was normally established in the anterior lateral plate mesoderm and the cardiac morphological defects are not apparent until the cardiac looping stage. tbx5 was expressed strongly both in the ventricle and the atrium in the defective hearts of D-glucose treated embryos other than the ventricle-enriched expression pattern observed in wild-type embryos. In contrast, tbx20 expression appeared dramatically lower throughout the heart. The bmp4 and notch lb expression was restricted to the myocardium and endocardium at the AV valve-forming region in wild-type hearts respectively. However, bmp4 expression was observed both in the myocardium and endocardium and the domains of expression greatly expanded, encompassing the part of the ventricle in the defective hearts. Likewise, notch 1b expression was observed both in the myocardium and endocardium and very faint expression appeared throughout the entire ventricle in the defective hearts. has2 expression was restricted to the valve-forming region in wild-type embryos while its expression was increased and the expression domain was significantly expanded throughout the AV boundary in the defective hearts of D-glucose treated embryos.Conclusions:Cardiac looping is affected earliest during heart organogenesis. The alteration of tbx5, tbx20 expression pattern in the defective hearts would contribute to the cardiac looping defects. The heart valve defects caused by elevated glucose are associated with the alteration of has2, notch1b and bmp4 expression pattern in the defective hearts.