Identification Of RyR2-PBmice And Effects Of Transposon Insertional Mutagenesis Of RyR2 Gene On Heart And Pancreas Function In Mice

Ryanodine receptors（RyRs）are the calcium release channels located in sarcoplasmic reticulum（SR）,which are composed of four subunits with a molecular weight 565 KD.RyRs can be divided into three subtypes: RyR1,RyR2 and RyR3,with different coding genes,respectively.RyR2 is mainly distributed in cardiac tissue,and participates in the excitation-contraction coupling of cardiac muscle through calcium-induced calcium release（CICR）.RyR2 plays an important role in maintaining the normal physiological activities of the heart,and its abnormal function can lead to arrhythmia,heart failure and other heart diseases due to the Ca²⁺ adjustment disorder caused by the abnormal RyR2.Transgenic animal model can be a vital instrument to discover and assess human diseases.The mouse genome highly resembles that of human,and the breeding cycle of mice is relatively short.Therefore,the mice are a kind of the most common model mammals.By transgenic technology,gene mutagenesis and other measures to change the sequence and structure of the related functional genes in mice,it is accessible to establish the animal models of human diseases,to explore the mechanism of disease development,and to develop therapeutic drugs and so on.Transposon is a DNA sequence that can change its position within a genome,and has been widely used in the studies of gene function.As a new transgenic carrier,transposon piggyBac（PB）is characterized by a high transposition activity,large load capacity,cutting without trace,and long-term stable inheritance,and it has been used to establish a mutant line-PBmice.RyR2 is an important intracellular Ca²⁺ releasing channel.This study aims to utilize the RyR2-PBmice,which is established by the model animal center of Fudan University,to explore the influence of PB insertion mutagenesis on the RyR2 functions and the biologic traits of mice.In the first part of the study,we screened out the heterozygous mice with piggyBac inserted into RyR2 gene（RyR2-PBmice）from the PB mutation database（PiggyBac Mutagenesis Information Center,http: //idm.fudan.edu.cn/PBmice/）.Among the RyR2-PBmice,the homozygotes exhibit intrauterine death and the heterozygotes grow as normal,which indicates that PB insertion mutagenesis could cause the loss or abnormality of RyR2 gene,and also reflects the significant function of RyR2 gene to sustain life in mice.We successfully identified the piggyBac translocated RyR2 gene heterozygous mouse by tracking the red fluorescent protein（RFP）carried by the transposable elements and PCR assay.Through the analysis of the growth status of the mice,it was found that the RyR2-PBmice can be stably inherited,and its growth,development and survival are not significantly different from those of wild-type mice（WTmice）.Because of the RFP sequence combined with the PB transposable elements,we detected the distribution of RFP with laser scanning confocal microscopy,and the distribution of RyR2 in vivo was monitored by immunofluorescence.The results showed that RyR2 was mainly expressed in the cardiac and skeletal muscle,smooth muscle and brain tissue.Compared with WTmice,there was no significant difference in the expression level of RyR2 in RyR2-PBmice.To further validate the expression level of RyR2 after transposition,the cardiac tissues from RyR2-PBmice and WTmice were subjected to RT-PCR and Western blot experiments at m RNA and protein levels,respectively.The mRNA level of RyR2 in RyR2-PBmice cardiac tissue was decreased significantly compared with WTmice,but the protein expression level of RyR2 has no significant difference between the two groups,indicating that RyR2-PBmice maintains the level of RyR2 expression comparable to WTmice by post-transcriptional modification,which may be an important reason why RyR2-PBmice can keep a normal cardiac excitation-contraction coupling and maintain normal growth.RyR2,a Ca²⁺ release channel in the sarcoplasmic reticulum of cardiomyocytes,is a key protein to maintain Ca²⁺ homeostasis and excitation-contraction coupling process in cardiomyocytes,as well as to keep systolic and diastolic function.Therefore,in the second part of the study,we first studied the effects of the piggyBac transposon insertion into RyR2 gene on the cardiac structure and function in RyR2-PBmice.The results showed that there are no significant differences in ECG features between RyR2-PBmice and WTmice when the two groups of mice in general anesthesia with isoflurane,no matter they are injected with epinephrine and caffeine or in the basal state,indicating that the PB insertion mutagenesis in RyR2 gene does not affect the heart function of mice at the basal state or at a stress state.It was remarkable that the sarcoplasmic reticulum Ca²⁺ capacity of cardiomyocytes reduced after caffeine stimulation according to the further measurement by laser scanning confocal microscopy,suggesting that the PB insertion mutagenesis in RyR2 gene has an effect on maintaining Ca²⁺ homeostasis in some degree,and the mild decrease of sarcoplasmic reticulum Ca²⁺ capacity could explain why RyR2-PBmice do not suffer arrhythmia.At the same time,it was observed that RFPs in the mitochondrion of cardiomyocytes were significantly higher than those in the cytoplasm under the laser scanning confocal microscopy,indicating that a large number of RFPs accumulated in the mitochondrion.It was shown that the mitochondrial ridge of cardiac tissue in RyR2-PBmice had a significant rupture and dissolution,and the structure of the mitochondrial membrane is incomplete by histopathological observation and transmission electron microscopy scanning.It was found that the potential of myocardial mitochondrial membrane in RyR2-PBmice decreased,the amount of ATP significantly reduced according to the mitochondrial function assessment.These results suggested that the piggyBac transposon insertion into RyR2 gene seriously affected the structure and function of the cardiac tissue mitochondrion in the mouse,leading to disorders of the energy metabolism.To further analyze the mechanism how the transposon insertion of piggyBac into RyR2 gene affects the myocytes,especially the mitochondrial structure and function of the mouse heart,we compared the transcriptome of RyR2-PBmice and WTmice and screened out parts of the differentially expressed proteins of the signal pathways closely related to the RyR2 and mitochondrial function.Besides,we used RT-PCR and Western blot analysis for further validation.We found that the piggyBac insertion mutagenesis in RyR2 gene has a significant impact on the RyR2 function of cardiomyocytes as well as the structure and function of mitochondrion,with the down-regulation of proteins associated with the respiratory chain,such as small GTP,cytochrome C oxidase,uncoupling protein（UCP）and mitochondrial calcium uptake protein.Interestingly,we found that the colour of the pancreas in the RyR2-PBmice biopsy was pinker than any other organs,and the boundaries with the surrounding tissues were obvious.Considering that RyR2 plays an important role in the regulation of insulin secretion and glucose in vivo,we studied the effects of the piggyBac insertion mutagenesis in RyR2 gene on the pancreatic structure and function in mice in the third part.Red particles in the pancreas of RyR2-PBmice are highly suspected to be caused by the enrichment of piggyBac transposon-carrying red fluorescent protein.Laser scanning confocal microscopy revealed that the pancreatic tissue was bright red.Although RFPs in the mitochondrion of the pancreas were less than those of the myocardium,it was found that RFPs of the pancreas were also accumulated in the mitochondrion with a more bright red than the surrounding tissue.The results of transmission electron microscopy showed that a lot of granules deposit in islet cells,which may be related to the redness of pancreas,and the mitochondrial ridge in RyR2-PBmice aslo had a significant rupture and dissolution.In the study on pancreatic function,we performed a glucose tolerance test in both RyR2-PBmice and WTmice mice.The results showed that the levels of plasma glucose and insulin were similar between the two groups.When glucose was stimulated,the plasma glucose level of RyR2-PBmice was significantly higher than that of WTmice,indicating that the ability of insulin resistance of RyR2-PBmice was significantly damaged.The immunoblotting results showed that the expression levels of the mitochondrial respiratory related proteins such as cytochrome oxidase C and uncoupling protein UCP were significantly decreased in the pancreas of RyR2-PBmice,while GLUT-4,which was directly involved in glucose transport,was close to that of WTmice.These results suggested that piggyBac transposon insertion into RyR2 gene did not directly affect the glucose transport of pancreatic tissue,but it could cause a metabolic disorder of the mitochondrial respiratory chain and indirectly affect the insulin secretion of pancreatic tissue.In conclusion,the animal model of homozygous transgenic mice was successfully established by inserting the piggyBac transposon carrying RFP into the RyR2 gene.The RyR2-PBmice exhibited intrauterine death while the heterozygotes survived well.Although it has not been found that the heterozygous mice（RyR2-PBmice）showed any abnormalities in heart function,it was revealed that the myocardial sarcoplasmic reticulum Ca²⁺ capacity decreased and the expression levels of mitochondrial respiratory chain-related proteins were significantly down-regulated,indicating that the insertion of piggyBac transposon into RyR2 gene caused a damage of mitochondrial energy metabolism.A large number of RFP deposits were found in the pancreatic tissue,and the secretion of insulin and the tolerance of glucose in RyR2-PBmice decreased.Similar to the study on cardiac structure and function,the expression levels of mitochondrial respiratory chain-related proteins in pancreatic tissue decreased,suggesting that the insertion of piggyBac transposon into RyR2 gene might lead to the decrease of pancreatic insulin secretion by affecting mitochondrial energy metabolism.