Establishment Of The Model Of Thyrotoxic Hypokalemic Periodic Paralysis Using CaV1.1-R528H Mutant Mice And Research On Its Mechanisms

Background and objectiveHypokalemic periodic paralysis mainly consists of sporadical hypokalemic periodic paralysis, familial hypokalemic periodic paralysis and thyrotoxic hypokalemic periodic paralysis. Familial hypokalemic periodic paralysis is a kind of related to abnormal skeletal muscle voltage-gated L-type calcium channel and sodium channel autosomal dominant genetic disease, mainly involved in the mutation gene of CACNA1 S,SCN4A and KCNE3, which CACNA1 S are the most common. For thyrotoxic hypokalemic periodic paralysis, the mutations of ion channel gene also have been found, such as KCNE3, KCNE4 and Kir2.6, but in addition to individual reports, the positive rate of less than 1%. At present, how the mutations of ion channel result in HPP or TPP is still not very clear. Related research of HPP at abroad has only limited changes in cell level and ion channel electrophysiology. In the early, we choosed Ca V1.1- Arg528His(R528H) mutations that was a good representation and had heavy symptoms, using homologous recombination and embryonic stem cell technology and successfully constructed Ca V1.1-R528 H mutant mice model. In our country, however,hypokalemic periodic paralysis are mainly composed of thyrotoxic hypokalemic periodic paralysis. In order to better study the pathogenesis of TPP, we further establish animal model of thyrotoxic hypokalemic periodic paralysis using Ca V1.1-R528 H mutant mice and study its mechanism. We expect to explore the related pathogenesis of Hypo PP from the whole animal level and to provide evidence for diagnosis and treatment of Hypo PP.MethodsPart 1 The identification of Ca V1.1-R528 H mutant mice8 Ca V1.1-R528 H mutant mice were randomly selected before the experiment, and were extracted genomic DNA from mice tail and were done its PCR identification. 5were randomly selected for DNA sequencing from 8 PCR products of genomic DNA of mice.Part 2 The comparison of biochemical indicators between Ca V1.1-R528 H mutant mice and wild-type C57BL/6J mice8-week-old Ca V1.1-R528 H mutant mice, each 8 male and female, and 8-week-old wild-type C57BL/6J mice, each 8 male and female were choosed. All mice started fasting, water deprivation at 20:00, and in the next morning 8:00 the eyeball blood were gathered in EP pipe and were inspected biochemical indicators, including 8indicators of glucose(GLU), alanine aminotransferase(ALT), aspartate aminotransferase(AST), creatine kinase(CK), serum creatinine(Cr) and electrolyte(Na +, K +, Cl-).Part 3 The discussion about the role of mutation factor, insulin and thyroxine on serum potassiumThirty-six 8-week-old CaV1.1-R528 H gene knock-in male mice and thirty-six8-week-old wild-type C57BL/6J male mice were chosen. Using three-factor two-level 2 × 2 × 2 factorial design( three factors included mutation, thyroxine and insulin, and two levels were with or without), the mice were divided into 8 groups.The thyroxine groups were injected levothyroxine(by 350μg/kg) intraperitoneally for12 days to prepare for the thyrotoxicosis status. Then the insulin groups were injected short-acting insulin(by 0.8U/kg) intraperitoneally after the last administration of levothyroxine, and the potassium level of different groups were measured and recorded before(0min) and after insulin injection(30min, 60min).Part 4 The pathogenicity role of FXYD1 protein in thyrotoxic hypokalemic periodic paralysisThe mice(wild + saline, mutation + saline, mutation + thyroxine + saline, mutation +thyroxine + insulin) were sacrificed after measuring the potassium, and immediately were taked the quadriceps of the hind legs, the size of about 1cm × 0.5cm × 0.2cm, and quickly were placed in 10% neutral formalin and fixed 24 h, and were paraffin-embedded. 4μm serial sections were dried at 65 ℃ incubator, and were stored at room temperature.We detected each group of mice FXYD1 protein expression in skeletal muscle by immunohistochemistry technique, and compared whether there were differences among different groups.ResultsPart 1 The identification of Ca V1.1-R528 H mutant micePCR and DNA sequencing results confirms that the mice are Ca V1.1-R528 H mutant mice, and DNA amplified fragment length of Ca V1.1-R528 H mutant mice are 870 bp,while the wild control group are 748 bp.Part 2 The comparison of biochemical indicators between Ca V1.1-R528 H mutant mice and wild-type C57BL/6J miceCa V1.1-R528 H mutant mice between male and female, and wild-type C57BL/6J mice between male and female are all no statistical difference(P > 0.05), and the comparison between groups with the respective gender also has no statistical difference(P > 0.05).Part 3 The role of mutation, thyroid hormone and insulin on serum potassium(1) Compared with the control group, the following phenomenon including irritability,dull coat, increased diet and water intake, and slowly increased body weight, was observed in the thyrotoxicosis mice. Thyroid function tests showed that the level of T3,T4 in thyrotoxicosis mice were significantly higher than the corresponding control mice( P < 0.05), and that TSH level was significantly lower than the corresponding control mice( P < 0.05).(2) After administration of insulin or thyroxine alone, there were no significant differences between mutant and wild-type mice in the potassium level, while after combined administration of thyroxine and insulin, there were a significant difference both at 30 min and 60min( P < 0.05), and the mutant mice was significantly lower the wild-type mice.(3) The main effects and interactions: Mutation factor or thyroxine factor alone did not work on the decrease of potassium level, and an effect of insulin were shown( P < 0.05); There were interactions between thyroxine factor and mutation factor, and between insulin factor and mutation factor( P < 0.05);There were no interactions between thyroxine factor and insulin factor.Part 4 The pathogenicity role of FXYD1 protein in thyrotoxic hypokalemic periodic paralysisThere are no statistically significant differences for FXYD1 protein expression levels between wild + saline group and mutation + saline group, and between mutation +saline group and mutation + thyroxine + saline group, and between mutation + saline group and mutation + thyroxine + insulin group(P>0.05).Conclusions Firstly, through the identification of mutant mice Ca V1.1- R528 H, we confirmed that the mutant gene is not lost in the passages; Secondly, A successful animal model of thyrotoxic hypokalemic periodic paralysis were established using Ca V1.1-R528 H gene knock-in mice; Thirdly, the subunit FXYD1 of Na~+, K~+-ATPase does not change in number in thyrotoxic hypokalemic periodic paralysis.