|Year : 2021 | Volume
| Issue : 4 | Page : 197-201
Gestational diabetes influences bone morphogenic protein signaling during heart development in C57BL mice
Fatemeh Ghasemzadeh1, Masoud Golalipour2, Kamran Haidari1, Zahra Nazari3, Mohammad Jafar Golalipour4
1 Department of Anatomical Sciences, Golestan University of Medical Sciences, Gorgan, Iran
2 Medical Cellular and Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran
3 Department of Biology, Faculty of Sciences, Golestan University, Gorgan, Iran
4 Gorgan Congenital Malformations Research Center, Golestan University of Medical Science, Gorgan, Iran
|Date of Submission||11-Dec-2019|
|Date of Acceptance||25-Jul-2021|
|Date of Web Publication||21-Dec-2021|
Prof. Mohammad Jafar Golalipour
Department of Anatomical Sciences, Gorgan Congenital Malformations Research Center, Golestan University of Medical Sciences, Gorgan
Source of Support: None, Conflict of Interest: None
Introduction: Gestational Diabetes Mellitus (GDM) is one of the most common metabolic complications of pregnancy that causes fetal mortality and morbidity. As uncontrolled gestational diabetes could induce congenital heart defects in the offspring. Therefore, this study was done to evaluate the effect of GDM on bone morphogenetic proteins (BMPs) gene expression during heart development in the C57BL mouse embryo. Material and Methods: In this experimental study, twelve 8-week old pregnant C57BL with an approximate weight of 130 g were randomly allocated into control and induced diabetic groups. On day 1 of gestation, the dams of the Diabetic group were received 150 mg/kg streptozotocin. While that of the control group were received an equivalent volume of normal saline. On day 11.5 of pregnancy, six embryos were withdrawn from each group. Total RNA was extracted from the cardiac tissue pieces of the embryos for expression of BMPs by quantitative real-time PCR. Results: BMP6 of the induced diabetic group increased to 2.4018-fold compared to the controls (P<0.05). While BMP 5,7, and 10 increased to (1.58, 1.0445, and 1.7623, respectively) and 1.7623-fold respectively in GDM in comparison to controls. Discussion and Conclusion: Therefore, it is suggested that the GDM could induce heart malformations by the upregulation of BMPs, particularly BMP6 expression.
Keywords: BMPs, gestational diabetes mellitus, heart
|How to cite this article:|
Ghasemzadeh F, Golalipour M, Haidari K, Nazari Z, Golalipour MJ. Gestational diabetes influences bone morphogenic protein signaling during heart development in C57BL mice. J Anat Soc India 2021;70:197-201
|How to cite this URL:|
Ghasemzadeh F, Golalipour M, Haidari K, Nazari Z, Golalipour MJ. Gestational diabetes influences bone morphogenic protein signaling during heart development in C57BL mice. J Anat Soc India [serial online] 2021 [cited 2022 Jul 3];70:197-201. Available from: https://www.jasi.org.in/text.asp?2021/70/4/197/333192
| Introduction|| |
Gestational diabetes mellitus (GDM), a type of Diabetes mellitus, affects approximately 7% of pregnant women, which is characterized by an impaired glucose tolerance during pregnancy.
On the other hand, Bone morphogenetic proteins (BMPs), a family of Transforming growth factor (TGF-βs) superfamily, is a heterodimer complex consisting of type I and II serine-threonine kinases and signals through the SMAD and non-SMAD pathways.,, BMP involves in some developmental processes homeostasis of tissues. BMPs are essential and play a main role in the formation of mesoderm, particularly myocardial development. van Gelder et al., a study has indicated that gestational diabetes is associated with an increased risk of prenatal mortality and major congenital anomaly. Some of the population-based studies have reported that the infants of diabetic mothers are more susceptible to complex diseases, including obesity, metabolic and cardiovascular complications during childhood and adolescence., It has been proven that children of mothers with GDM have more complex congenital heart anomalies and it seems that, hyperglycemia is a risk factor that has adverse impacts on the development of the cardiovascular system.,There are few articles about the molecular mechanisms of congenital cardiovascular malformation due to gestational diabetes.
BMPs have crucial roles in cell proliferation, differentiation, migration, and apoptosis during organ development and adult life. As their inductive roles cardiac differentiation heart morphogenesis. Also, BMPs are pivotal in the regulation of septovalvular development during the formation of heart chambers. In the heart tissue, some of BMPs are expressed before cardioblast and throughout the later stages of heart development. It has been clear that the main roles of six BMPs (BMP 2, 4, 5, 6, 7, and 10) are expressed in the heart during development.
Despite an understanding of the effects of BMPs on cardiac development, but we were wondering whether GDM has any impacts on the expression of BMPs during development.
On days 7.5 and 8.5, the BMP2 level is prominent on day 7.5 in cardiac crescent pro-myocardium and around mesoderm cells, while lower levels are in the outflow tract on day 9.5. The BMP2 expression in the AV channel is turned off on day 10.5. BMP4 in the Outflow tract on day 8.5 and BMP57 is highly expressed in the developing heart, as BMP6 is expressed in the myocardium and atrial-ventricular cushions of the outflow tract (OFT). But it in AV (Atrioventricular) and OFT (Outflow tract) pads disappear on 9.5 and reduces on the right OFT on 10.5 and the 11.5. BMP10 is expressed initially in all heart chambers of a rat. The role of BMPs as a signal involved in septation, as any defect in this process will be one of the most common causes of congenital heart disease. Therefore, signaling pathways of BMP in mesoderm formation and cardiac development is vital. BMP signaling regulates the induction of cardiac differentiation through a series of genes involved in the cardiogenesis by expression of NKX2.5 and GATA4 transcription factors. That is why this study is designed to investigate the effect of STZ-induced gestational diabetes with expression changes of BMP5, BMP6, BMP7, and BMP10 in the heart tissue of E11.5 C57BL mice embryo.
| Material and Methods|| |
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Golestan University of Medical Sciences, Gorgan, Iran (IR.gums.REC.1395.155)
Generation of gestational diabetes mellitus model
Twelve, 8-week old C57BL mice with an approximate weight of 130 g were housed separately with a male overnight for copulation. The next day vaginal plaque is checked, so it is seen considered as day 0 of pregnancy. Pregnant mice were allocated randomly into control and diabetic groups. Diabetes induced by a single intraperitoneal injection of a freshly prepared streptozotocin solution (150 mg/kg) in saline normal (0.85%) on day 1 of gestation. Dams of the control group were injected with an equal volume of saline normal. 72 hours later, blood glucose levels were checked using a glucometer (ACCU CHEK, Roche Diagnostics, Germany). And the mice with a plasma glucose level higher than 200 mg/dl, was considered as the GDM model. Also, the blood glucose level of the control group was checked before and after pregnancy.
Heart tissue collection
Control and diabetic mice were sacrificed by cervical dislocation on day 11.5 of gestation. Then, the embryos were removed by cesarean section. Heart tissues were extracted from embryos under a stereomicroscope and stored at −80°C until further use.
RNA cDNA synthesis
Total heart tissue from embryos of diabetic and control groups used for RNA extraction. Briefly, RNA was isolated from heart tissues using TRIZOL (Invitrogen) reagent according to the manufacturer's protocol. Residual DNA was digested with 10 U RNase-free DNase (TaKaRa) in the presence of 20 unit RNase inhibitor. After heat inactivation, the total RNA solution was removed and kept at −80°C. The concentration and purity of total RNA samples were measured using a Picodrop Spectrophotometer. The stability and integrity of the RNAs were estimated by agarose gel electrophoresis. For reverse transcription, 1 μg of total RNA was amplified using a cDNA synthesis kit (Fermentas).
Real-time reverse transcription-polymerase chain reaction assay
The forward and reverse polymerase chain reaction (PCR) primers for the 7 genes (S18, BMP 5, 6, 7, and 10) were designed using primer 3 software and were synthesized by Metabion (Martinsried, Germany). All the primer sequences are listed in [Table 1]. Real-time PCR was performed using the SYBR-Green PCR Master Mix kit (TaKaRa) in the Thermo Cycler (ABI, 7300). Mouse S18 used as a housekeeping gene and cDNA from the control group used as a calibrator. The relative gene expression level between the two groups was determined by the comparative cycle threshold (CT) method. The specificity of the amplified product was confirmed by gel electrophoresis. Every Real-time PCR experiment was repeated with four samples and each sample was run in duplicate.
|Table 1: Real-time polymerase chain reaction primer name, sequences, size, and GenBank accession number|
Click here to view
The value of 2-ΔΔCT was used to calculate relative changes in gene expression. Amplification plot comparison and CT comparison (CT, the first cycle with fluorescent intensity greater than baseline) are estimated for each gene. Finally, relative mRNA expression levels among diabetic and nondiabetic groups were determined by the CT method. Fold change gene expression was shown. Relative target gene expression (fold change) and blood glucose level data were analyzed by one-way ANOVA using Rest statistical analysis software. The differences between the two groups were compared using Student's t-tests and all P values were considered statistically significant If P < 0.05. Data were presented as a mean ± standard deviation.
| Results|| |
Blood glucose level
Fasting blood glucose was measured in both control and diabetic group on day zero (G0) and day 3 (G3) of gestation. In diabetic pregnant mice, blood glucose level was significantly increased 72 h after STZ injection (P < 0. 05). The mean ± standard error of mean of blood glucose concentrations in control and diabetic mice is depicted in [Figure 1].
|Figure 1: Serum glucose concentrations in diabetic and control pregnant mice on the zero-day of pregnancy (G0) and the third day of pregnancy (G3). Values are means ± standard error of mean. **P < 0.01|
Click here to view
Quantitative Reverse Transcription-polymerase chain reaction results
In this study, we assessed whether the expression levels of bone morphogenetic genes including BMP5, BMP6, BMP7, and BMP10 were affected by gestational diabetes in the heart tissue of the embryo. Our data showed that the mRNA expression levels of all studied genes were higher in the heart tissue of embryos derived from GDM compared to controls. Data analysis showed that there was a significant increase in BMP6 in the heart tissue of the GDM-derived embryos in comparison with controls (**P ≤ 0.005). Although GDM-derived embryos showed increases in expression of BMP 5, 7, and 10, no significant differences were seen in compared to controls [Figure 2].
|Figure 2: Real-time reverse transcription-polymerase chain reaction analysis of BMP-5, -6, -7, and-10 mRNA expression in the hearts of control and diabetic-derived embryo. Bar graph representing the fold changes of mRNA levels quantified by normalizing to the S18 as a housekeeping gene. Data are presented as mean ± standard deviation. BMP: Bone morphogenetic protein|
Click here to view
| Discussion|| |
Diabetes in pregnant women is associated with a fivefold increase in the risk of cardiovascular malformations in their offspring. The most common congenital heart defects are associated with cardiomyocyte hypertrophy, interventricular septum defects, common outflow tract, and heart valves malformations., In laboratory animals, it has been proven that the thickness of the ventricular septum and the weight of the heart in the Offspring of STZinduced diabetic rats is significantly higher than untreated Ones.,BMP is involved in the regulation of many processes underlying cardiovascular development., Therefore, the researchers show that BMP signaling has an important role in heart morphogenesis after the midgestation stage.,
This study was designed to determine the effect of induced GDM on BMPs expression in the heart tissue of E11.5 embryos in C57BL mice. In the present study, the expression of all studied genes in the E11.5 embryos of C57BL diabetic mice was higher than controls. We observed more than a 2-fold increase in BMP6 in the heart tissue of mouse embryos obtained from diabetic mothers (**P ≤ 0.005). Furthermore, we also showed that GDM upregulates expression of BMP10 and BMP5 in embryonic heart tissue by 1.76 and 1.58 fold, respectively.
Previous studies demonstrated that the BMP signaling pathway regulates the differentiation of cardiomyocytes from the mesoderm. Although, many studies have shown the role of BMPs in the formation of the ventricular chambers and septovalvulogenesis plus. On the other hand, both inhibition and stimulation of BMP are required for normal cardiomyocyte differentiation during heart development., So, these proteins can play a dual role in heart development.
In the developing heart under hyperglycemia, it seems that oxidative stress associated with malformations and subsequently formation of free radicals disrupt TGF-β and Wnt signaling. Possible pathways concerning GDM induced congenital heart defects have occurred through BMPs shown in [Figure 3]. BMPs form a complex with type I and type II receptors, and the type I receptor causes phosphorylation of SMAD1, SMAD5, or SMAD8. Phosphorylated SMADs form a complex with SMAD4. Which is transported into the nucleus and affects the genes GATA4, NKX2/5, and MEF2C, which play an important role in normal heart development.
|Figure 3: Possible pathway for gestational diabetes mellitus-induced congenital heart defects through bone morphogenetic proteins|
Click here to view
| Conclusion|| |
Despite the importance of the BMPs in cardiac development and also the influence of GDM on the induction of heart malformations, no detailed study has been conducted on the effect of maternal diabetes on the expression of these genes in developing a heart. In conclusion, our data indicated that GDM can cause the upregulation of BMPs in the developing heart of E11.5 embryos in C57BL mice. Upregulated BMPs can affect important genes involved in cardiomyocyte hypertrophy. Taken together, these findings might be helpful in the understanding of the mechanism underlying GDM caused heart failure in the offspring. However, it seems that studying normal and abnormal cardiac development is necessary to fully appreciate the exact molecular mechanism of inducing cardiac defects in offspring by gestational diabetes.
The developmental period of the heart occurs on different days in uterus (7.5–15.5), but in this study due to the limitations, only on day 11.5 of gestation. Furthermore, no histological and morphological study was performed on the tissue surface, only at the level of gene expression has been examined.
This study was funded by Golestan University of Medical Sciences (grant number: 174411). Hence, we acknowledge the support of Golestan University of Medical Sciences for financial support of this research.
Financial support and sponsorship
This study was financially supported by Deputy of research of Golestan University of Medical Sciences, Gorgan, Iran.
Conflicts of interest
There are no conflicts of interest.
| References|| |
American Diabetes Association. 2. Classification and Diagnosis of Diabetes. Diabetes Care. 2015;38(Supplement 1):S8.
Dennery PA. Effects of oxidative stress on embryonic development. Birth defects research Part C, Embryo today : reviews. 2007;81:155-62.
Miyazono K, Kamiya Y, Morikawa M. Bone morphogenetic protein receptors and signal transduction. Journal of biochemistry. 2010;147:35-51.
Wang J, Greene SB, Martin JF. BMP signaling in congenital heart disease: New developments and future directions. Birth defects research Part A, Clinical and molecular teratology. 2011;91:441-8.
Bandyopadhyay A, Yadav PS, Prashar P. BMP signaling in development and diseases: A pharmacological perspective. Biochemical pharmacology. 2013;85:857-64.
Wang RN, Green J, Wang Z, Deng Y, Qiao M, Peabody M, et al. Bone Morphogenetic Protein (BMP) signaling in development and human diseases. Genes & diseases. 2014;1:87-105.
Van Gelder MM, Van Bennekom CM, Louik C, Werler MM, Roeleveld N, Mitchell AA. Maternal hypertensive disorders, antihypertensive medication use, and the risk of birth defects: a case-control study. BJOG: An international journal of obstetrics and gynaecology. 2015;122:1002-9.
Correa A, Gilboa SM, Besser LM, Botto LD, Moore CA, Hobbs CA, et al. Diabetes mellitus and birth defects. American journal of obstetrics and gynecology. 2008;199:237.e1-9.
Mitanchez D, Yzydorczyk C, Siddeek B, Boubred F, Benahmed M, Simeoni U. The offspring of the diabetic mother--short- and long-term implications. Best practice & research Clinical obstetrics & gynaecology. 2015;29:256-69.
Chu C, Gui YH, Ren YY, Shi LY. The impacts of maternal gestational diabetes mellitus (GDM) on fetal hearts. Biomedical and environmental sciences : BES. 2012;25:15-22.
Wang G, Huang WQ, Cui SD, Li S, Wang XY, Li Y, et al. Autophagy is involved in high glucose-induced heart tube malformation. Cell cycle (Georgetown, Tex). 2015;14:772-83.
Piotrowska I. Functional implications of Bone Morphogenetic Protein 10 (BMP10) expression in pathological hearts: Max Planck Institute for Heart and Lung Research, Bad Nauheim. Justus-Liebig university of Giessen; 2007.
Harmelink C, Jiao K. Bone Morphogenetic Protein Signaling Pathways in Heart Development and Disease. Congenital Heart Disease 2012.
Association AD. Diagnosis and Classification of Diabetes Mellitus. Diabetes Care. 2013;36(Supplement 1):S67-S74.
van Wijk B, Moorman AF, van den Hoff MJ. Role of bone morphogenetic proteins in cardiac differentiation. Cardiovascular research. 2007;74:244-55.
Damasceno DC, Sinzato YK, Bueno A, Netto AO, Dallaqua B, Gallego FQ, et al. Mild diabetes models and their maternal-fetal repercussions. Journal of diabetes research. 2013;2013:473575.
Lister R, Einstein F, Chamberlain A, Dar P, Bernstein P, Zhou B. Streptozotocin dosing for the induction of fetal cardiac dysmorphology associated with maternal hyperglycemia. American Journal of Obstetrics & Gynecology. 2012;206:S131.
Zielinsky P, Luchese S, Manica JL, Piccoli AL, Jr., Nicoloso LH, Leite MF, et al. Left atrial shortening fraction in fetuses with and without myocardial hypertrophy in diabetic pregnancies. Ultrasound in obstetrics & gynecology : the official journal of the International Society of Ultrasound in Obstetrics and Gynecology. 2009;33:182-7.
Corrigan N, Brazil DP, McAuliffe F. Fetal cardiac effects of maternal hyperglycemia during pregnancy. Birth defects research Part A, Clinical and molecular teratology. 2009;85(6):523-30.
Menezes HS, Zettler CG, Calone A, Correa JB, Bartuscheck C, Costa CS, et al. Regression of gestational diabetes induced cardiomegaly in offspring of diabetic rat. Acta cirurgica brasileira. 2009;24:251-5.
Reinking BE, Wedemeyer EW, Weiss RM, Segar JL, Scholz TD. Cardiomyopathy in offspring of diabetic rats is associated with activation of the MAPK and apoptotic pathways. Cardiovascular Diabetology. 2009;8:43.
Yu W, Zha W, Guo S, Cheng H, Wu J, Liu C. Flos Puerariae extract prevents myocardial apoptosis via attenuation oxidative stress in streptozotocin-induced diabetic mice. PloS one. 2014;9:e98044.
Guo WT, Dong DL. Bone morphogenetic protein-4: a novel therapeutic target for pathological cardiac hypertrophy/heart failure. Heart failure reviews. 2014;19:781-8.
Caliceti C, Nigro P, Rizzo P, Ferrari R. ROS, Notch, and Wnt signaling pathways: crosstalk between three major regulators of cardiovascular biology. BioMed research international. 2014;2014:318714.
[Figure 1], [Figure 2], [Figure 3]