Establishment of the SCH rat model
Establishment of the SCH rat model refers to the previously literature .The rats were injected with 3% pentobarbital sodium (0.1 mL/100 g) and underwent thyroidectomy, while Sham group rats underwent sham thyroid surgery. The rats were fed normally for 4 w after operation, then the blood was collected from the retroorbital venous plexus, and serum TSH and TT4 were detected. When serum TSH levels were higher than that in Sham group, the TT4 levels were lower than that in Sham group, confirming the successful establishment of the SCH rat model. Four weeks after surgery, rats in the SCH group were injected subcutaneously with L-thyroxine (L-T4, Sigma, USA) 1.0 µg/100 g/day on the neck. Sham group rats were injected subcutaneously with physiological saline (50 µL/100 g/day) on the neck. Calcium lactate (0.1% w/v) was added to the drinking water for all rats after surgery. Nine days later, all rats were mated with normal male rats (male: female = 1: 2). The pregnant rats were then kept in single cages until delivery. The day of vaginal plus was confirmed by microscopic observation and designated as E0. Serum and tissue samples were collected at E16, E18, P5 and P10. At the end of the experiment, all rats were anesthetized with pentobarbital (50 mg/kg, intraperitoneal) and euthanized by thoracotomy and hearts removal.
Measurement of TT4 and TSH
Blood samples obtained from the rats were immediately centrifuged at 13,000 g for 15 min and stored at -80°C for measurement of serum TT4 (#CEA452Ge, Youersheng, China) and TSH (#CEA463Ra, Youersheng, China) using a supersensitive chemiluminescence immunoassay.
ATPase activity analysis
The ATPase activity was detected using an ultramicro Ca2+-ATPase kit and Na+/K+-ATPase kit (#A070, Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. The succinate dehydrogenase (SDH) activity was measured by kit(A022, Nanjing Jiancheng Bioengineering Institute, China). Protein content was measured with a Coomassie blue protein assay kit (#WLA004a, wanleibio, China). ATPase activity was expressed as mol Pi liberated per mg protein per hour (mol Pi/(mg prot·hr)).
RNA isolation and quantitative real-time PCR
RNA was extracted from heart tissues from each group using TRIzol (Life Tech). First strand of cDNA was synthesized using total RNA and RT-PCR was carried out by TaqMan expression assays, and β-actin (#WL01845, wanleibio, China) was used as a internal reference. The sequences for Nkx2-5,Gata4༌BMP4༌Smad4 and GAPDH were performed through the ABI PRISM system. Primer sequences are shown: Bmp4 F 5’-ATCGTTACC TCAAGGGAGTGGA-3’; Bmp4 R 5’-ATCGTTACCTCAAGGGAGTGGA-3’; Samd4 F 5’-CGTTCACGAGGCATTTAC-3’; Samd4 R 5’-GGGAGGGAGTTGGACTG-3’; Nkx2-5 F 5’-TGGACAAAGCCGAGACAGAC-3’; Nkx2-5 R 5’-TCAGCGGGCGACAGGTA-3’; Gata4 F 5’-AAACGGAAGCCCAAGAAT-3’; Gata4 R 5’-GCTGCTGTGCCCATA GTGAG-3’; β-actin F 5’-GGAGATTACTGCCCTGGCTCCTAGC-3’; β-actin R 5’-GGCCGGACTCATCGTACTCCTGCTT. Reactions were performed in a total volume of 20 µL and gene expression was determined by SYBR Premix Ex Taq TM II (TaKaRa Biotechnology Co., Ltd.) in accordance with the manufacturer’s instructions. Reactions began with a 10 s hot activation of Taq polymerase at 95 °C, followed by 40–45 cycles of amplification in three steps (denaturation at 95 °C for 5 s, 30 s annealing at 60 °C and 30 s extension at 72 °C). The mRNA expression was measured as a ratio to β-actin.
Immunohistochemistry staining was performed to localize Nkx2-5 (1:500, #A12688, wanleibio, China), Gata4 (1:1000, #WL01293, wanleibio, China), BMP4 (1:500, #WL02806, wanleibio, China) and Smad4 (1:300, #WL02049, wanleibio, China). Heart tissues were routinely embedded in paraffin, and sectioned at a 3–5 µ m thickness. All sections were dewaxed, and incubated with 0.3% H2O2 for 10 min. The antigen was repaired by heating 0.03M citrate buffer (pH6.0) for 40 min at 95 °C, and then incubated with 5% goat serum albumin for 20 min to block the non-specific binding sites. All sections were incubated with primary antibody overnight at 4 °C in a wet box and stained with sheep anti-rabbit IgG-HRP secondary antibodies (Abcam, UK). The sections were stained using 3,3-diaminobenzidine (DAB) (Sigma, USA) and counterstained with hematoxylin solution. Tissue sections without primary antibodies were the negative controls. Finally, samples were observed and photographed under a microscope.
Western blotting was performed as described previously . Heart samples were lysed in complete RIPA buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride and 1 × protease inhibitor cocktail [Roche]) and homogenized using a Sonic Dismembrator 100. The concentration of tissue protein was detected using a BCA protein concentration test kit(#WLA004, wanleibio, China), and equal amounts of soluble protein were separated on 10% polyacrylamide gels, transferred onto a nitrocellulose membrane, and followed by routine western blot analysis. Primary antibody: Nkx2-5 (1:500, #A12688, wanleibio, China), Gata4 (1:1000, #WL01293, wanleibio, China), BMP4 (1:500, #WL02806, wanleibio, China) and Smad4 (1:300, #WL02049, wanleibio, China). Proteins were visualized using a ClarityTM Western ECL Substrate (#WLA003; wanleibio, China) and a Tanon 5200 Full automatic chemiluminescence image analysis system (Tanon Science and Technology Co., Ltd., Shanghai, China).
Statistical analysis was performed using SPSS 20.0 statistical software (IBM Corp., Armonk, NY, USA). Variables are expressed as the means ± standard error of the mean (SEM). Data were analyzed using t-tests and one-way ANOVA. Statistical significance was reached at a two-sided p < 0.05.