Assess Hypokalemia Disease Models Of Cchl1a3-R528H Gene Knock-in Mice And Explore Its Mechanism

Background and objective Cchl1a3 gene(corresponding to human CACNA1 S gene)section 528 arginine replaced by histidine(R528H) is a missense mutations related with Hypo PP.Hypo PP is a group of periodic recurrent flaccid skeletal muscle weakness or muscleparalysis neuromuscular diseases characterized by abnormal ion channels,pathogenesis of this disease is not very clear,generally considered related with the serum potassium concentration of intracellular fluctuations.In recent years,studies have found that the patient’s own Hypo PP carrying the mutant gene,mainly involved in ion channel mutations including CACNA1 S,SCN4A and KCNE3,they are encoded in skeletal muscle L‐ type voltage‐gated calcium channel α1 subunit Ca V1.1, sodium channel α subunit Na V1.4 and potassium channel auxiliary subunit Mi RP2.CACNA1S‐R528H mutation is most common.Currently researches related to Hypo PP only confined cellular level and electrophysiological changes of ion channels,how associated mutations leading to hypokalemia and periodic paralysis did not make a satisfactory explanation.Our group select a representative prominent Ca V1.1‐R528H mutation,applicate Red/ET technology and FLP/frt site‐specific recombination system successfully constructed Cchl1a3‐R528H gene knock‐in mice model,In this study, assess if the model can be used as animal models of disease to research Hypo PP and explore its mechanisms,so that, to explore the pathogenesis of Hypo PP from the whole animal level and to provide evidence for clinical treatment of Hypo PP.MethodsPart1 Detect some biochemical indicators of Cchl1a3‐R528H gene knock‐inmice and verify dry chemical assay rat tail blood electrolytes feasibility:select 8‐week‐old wild‐type C57BL/6J mice 20(half male half female)and 8‐week‐old Cchl1a3‐R528H mutant knock‐in mice 20(half male half female),detect aspartate aminotransferase(AST),alanine aminotransferase(ALT),glucose(GLU),serum creatinine(Cr),blood electrolytes(K+,Na+,Cl‐) and a number of other biochemical indicators,and use dry chemical assay tail blood and vein blood electrolytes(K+,Na+,Cl‐)of Cchl1a3‐R528H mutant gene knock‐in mouse,compare with each other. Part2 Explores the different doses of epinephrine effecting serum potassium of Cchl1a3‐R528H mutant gene knock‐in mouse and its related mechanisms:select 8‐week‐old male wild‐type C57BL/6J mice and 8‐week‐old male Cchl1a3‐R528H mutant gene knock‐in mice each 40,after randomization,by the way of intraperitoneal injection,respectivelyinject saline,epinephrine,and U0126(the MEK inhibitor),were recorded serum potassium before injection 0min(baseline value),10,20,30,40,50,60 min after injection.Results Firstly,biochemical indicators of Cchl1a3‐R528H mutant gene knock‐in mice was no significant difference between the wild‐type C57BL/6J mice(P>0.05), wild‐type C57BL/6J mice can be a good reference specimens in subsequent experiments; compared dry chemical method measuring the tail blood electrolytes with wet chemical method the measuring blood electrolytes,there was no significant statistical difference(P>0.05),demonstrated the feasibility of a dry chemical method measuring the rat tail blood electrolytes.Secendly,two control group after injecting saline,each time period before and after the experiment,no significant changes in serum potassium(P>0.05);and after injection of different doses epinephrine,both in wild‐type C57BL/6J mice or in Cchl1a3‐R528H mutant gene knock‐in mice,have led to decreased serum potassium levels in mice.The same dose group,serum potassium of Cchl1a3‐R528H mutant gene knock‐in mice reduced significantiy compared withwild‐type C57BL/6J mice, difference was statistically significant(P<0.05);comparison of different dose groups,the difference was not statistically significant(P>0.05),immeasurable‐effect relationship; secondary interactions of dosage and whether mutation was also not statistically significant(P>0.05).After the MEK inhibitor U0126 treatment,and then injected the epinephrine group,found all the time before and after the test section of mouse serum potassium was no significant change(P>0.05),and potassium of pure injecting epinephrine group decreased significantly,compared the two groups,the difference there was statistically significant(P<0.05).Conclusions Firstly,the present study has verified the hypokalaemic effects of adrenaline, and the most significant decline in 20min;thenverify the feasibility of using a dry chemical method to repeat testing micro rat tail blood electrolytes,provide a foundation for our experiments and a reference to the other similar experiments; confirmed Cchl1a3‐R528H mutant gene is pathogenic of hypokalemic periodic paralysis;confirmed Cchl1a3‐R528H knockin mice as hypokalemic periodic paralysis disease animal model was successful;demonstrated by the level of the whole animal adrenaline in skeletal muscle cells are active Na+ pump activity by ERK signaling pathway to reduce serum potassium. for the first time.