The Connexin43and45Expression In The Cardiocytes Of Mice Congential Hypothyrodism Model

Background:Thyroid hormone (TH) plays a key role in the growth and differentiation of many organs. Congenital hypothyroidism (CH), a common pediatric endocrine disease, is a main cause of childhood mental retarded. Beside mental retarded, dysfunctions of many other organs were reported in paients with CH. Heart disorders, including congenital heart diseases, abnormal fetal heart rate tracings, congenital heart block, arrhythmia, even ventricular function, were involved frequently. Moreover, some studies showed that levothyroxine replacement therapy can improve the ventricular function, arrhythmia, and even closure of the ductus arteriosus.The mechnisim of heart disorders in CH patients is still need further study. TH binds its receptor (TH receptor, TR) in the nucleus, which plays a role on regulation the target gene transcriptional level by recognition the thyroid hormone response elements (TREs) in the promoter. Some studies showed that CH may affect some genes expression in heart. However, the candidate genes which be regulated by TH and result in heart disorders are still unclear.Gap junction (GJ) is the morphological substrate of the type of electrical synapse and mediates the GJ communication (GJC) among adjacent cells. GJ is composed of a pair of connexon that includes6connexin (Cx) proteins and make a hemichannel with a diameter of about1.5nm. There are abundant Cx in the heart, including Cx43, Cx45and Cx40. Cx43is the predominant type and accounts for90-95%of the total Cx. These Cxs play important role on the heart development and physiological function. They mediate the spread of the electrical impulse which triggers synchronized contraction of the cardiac chambers and contributing to the coordination of activities between cells of the arterial wall. Although several studies showed that increased TH may affect the Cx43expression on the cardiocytes, the results are still controversial. Moreover, no similar study about Cx43and Cx45in hypothyroidism condition was reported.Herein, we aim to investigate the effect of TH on Cxs expression by measuring the Cx43and Cx45levels in cardiocytes of mice CH model.Objectives:To investigate the effect of thyroid hormone (TH) on connexin (Cx) expression by measuring the Cx43and Cx45expression in cardiocytes of mice congenital hypothyroidism (CH) model.Materials and Methods:Animal ModelHealth adult C57BL/6J mice mated together. They were divided into CH group and control group according to the different intervention. The control group is definited as feeding of clean drinking water throughout. The CH group is definted as feeding0.03%thiamazole contained water from the10th day after mate to7th day after the offsprings’ birth. Their offsprings were sacrificed on day1,7, and14. Saline perfusion was performed and the heart (ventricle) was collected for study. Real time RT-PCRThe levels of Cxs mRNA were measured by real time RT-PCR. Total RNA was extracted using AxyPrep Mutilsoure Total RNA Miniprep Kit (Axygen Biosciences. USA). Self-designed primers and SYBR PrimeScriptTM PCR Kit (TaKaRa Bio Group, Dalian, China) were used real time RT-PCR. The sequences of the primers used in this study were shown in Table1. Two-step method was used for Cx43and Cx45. Thermocycling conditions consisted of40cycles of94℃for15s and63℃for35s, preceded by an initial denaturation step at94℃for3min. After amplification, melting curve analysis was done to verify the correctness of the amplicon.△Ct was presented for target gene mRNA levels. A negative control without cDNA was run with every PCR to assess the specificity of the reaction.ImmunohistochemistryThe tissue was fixed with10%buffered neutral formalin. Unstained4μm sections were cut from each tissue microarray and embeded in paraffin. Sections were stained immunohistochemically using the ChemMateTM Dako EnVisionTM two-step system (HRP). The rabit anti-Cx43antibody was obtained from Labvision Neo-Markers (Westinghouse, USA) and rabit anti-Cx45antibodies were obtained from Santa Cruz Biotechnology Inc (USA). The secondary sheep anti-mouse or anti-rabitt IgG antibodies were purchased from Beyotime Institute of Biotechnology (Jiangsu, China).Results:Model CheckingBlood was obtained when the pups were sacrificed for thyroid hormone level analysis. We found that T4and free T4levelson day7and14in the CH group were lower than these in controls. The effect of CH on the expression of Cx43and Cx45In control group, the levels of Cx43mNRA on the cardiocytes decreased after birth and increased from day7. However, the Cx43mRNA levels in CH group were not increased after day7. Compared with the control group, the Cx43mRNA levels in CH group were significantly lower on day7and14(P＜0.001, respectively). Immunohistochemistry showed that the Cx43protein were abundant on the intercalated disc in both groups, especially in the controls.The Cx45mRNA increased after birth in both groups. There were not significantly different between control and CH group on3time points (all P>0.05) although the△Ct was slight higher in CH group. Immunohistochemistry showed abundant Cx45protein on the intercalated disc in both groups without significant difference.Conclusion:1. Low TH levels decrease the expression of Cx43in the cardiocytes of mice, which may be a reason of heart disorder in CH patient.2. The Cx45mRNA levels have not statistical difference between the control and CH groups, which implied the expression of Cx45independence with TH during these period.