The Role And Mechanism Of New Mutation Of AMPK Gamma 2 Subunit In PRKAG2 Cardiac Syndrome

Background: The main clinical phenotypes of PRKAG2 Cardiac Syndrome are cardiac hypertrophy, ventricular pre-excitation and the progressive lesions of cardiac conducting system(CCS). It is a rare autosome dominant disease and is mainly caused by the mutation of PRKAG2 gene which belongs to AMPK γ2 subunit of adenosine monophosphate.AMPK widely exists in the tissue of mammals and is named “regulator of cellular energetics”. The balance of cellular energetics inside can be realized through the regulation of glucose and lipid metabolism. AMPK are heterotrimeric proteins which are composed by catalytic subunit α, regulatory subunit β and γ. The γ2 subunit contains 4 cystathionine β-synthase(CBS) areas which are made up of 60 aminoacid. The tandem between them then formed Bateman domain.Bateman domain, together with the capacity changes of AMP, can cause the active changes of AMPK and then change the metabolism and balance of cellular energetics. Up to now, it is reported internationally that no less than 20 PRKAG2 cardiac syndrome families located on AMPK γ2 subunit mutation of autosome 7q3 and there are 11 PRKAG2 gene mutation in all(including 1 frame-shift mutation and 10 missense mutation). A further sequencing shows that: all the mutations place centrally over the CBS area of γ2 subunit, which decreases the combining capacity of Bateman domain and AMP. It can result in such typical PRKAG2 cardiac syndrome symptoms. In 2007, Doctor Zhang Jing from Cardiology Department of Changhai Hospital reported firstly about the typical PRKAG2 cardiac syndrome family of the Hans and tracked gene sequencing. This family has the same general characteristics as the family reported by foreign countries, including cardiac hypertrophy, pre-excitation syndrome, advanced atrioventricular block, sinus node dysfunction, sudden death and atrial arrhythmia such as atrial flutter and atrial fibrillation. However, there are some differences from CBS areas mutation that reported formerly. During the sequencing, it can be discovered that the G100 mutation site locates on exon 3 outside CBS area. But the foreign reports shows all sites sequencing are completely normal. Here are some questions: Whether the G100 S mutation outside CBS can cause the same change of AMPK function as the CBS area mutation site do? What’s the comparison between the biology effect of AMPK function change and the mutation inside CBS area? Therefore, the prophase research group has done the relevant studies, showing that the gene mutation can cause the change of the action potential on vitro cell level. The studies also discussed preliminarily about the mechanisms of arrhythmia induced by the mutation. They also demonstrated that G100 S mutation could lead to cardiac hypertrophy on fish level and then discussed about the relevant mechanisms. Considering that zebrafish model can not be checked under the electrocardiogram examination, so the diagnosis of arrhythmia can not be done from the symptoms. Consequently, there are still deficiencies in arrhythmia research currently. Thus the transgenic mouse model has incomparable advantages both in further study of G100 S mutation in vivo and in pathogenic mechanism. The research group studies intensively by building mammals transgenic models which approach to genetic homology of human beings. For the transgenic mouse models of over expression PRKAG2(G100S) mutations, a further study can be done through the observation of the symptoms phenotypes and the mechanisms this time. It is helpful for deepening the comprehension of the AMPK γ2 subunit functions outside CBS areas. This can be crucial and valuable in comprehensive study of clinical phenotypes and the mechanisms of the mutation site in PRKAG2 cardiac syndrome families of the Hans. All of the above studies may provide new ideas in treating PRKAG2 cardiac syndrome which is typically exists among Chinese.Purpose: By building PRKAG2(G100S) transgenic mouse models, phenotypes such as myocardial hypertrophy and arrhythmia can be observed. So the study can make further discussions and do researches on pathogenic mechanism of myocardial hypertrophy and arrhythmia caused by the new G100 S mutations in PRKAG2 cardiac syndrome.Methods:(1) The structure of PRKAG2(G100S) transgenic mouse models: mouse Myh6(α-MHC) gene promoter of cardiac-specific expression is chosen to drive the expression of PRKAG2(G100S). PRKAG2(G100S) fragment, Myh6(α-MHC)Promoter and poly A can be connected through molecular cloning. The insulator component can be added on the two sides for the use of stable expression. The outermost endonuclease of transgenic fragment is used to release transgenic fragments. By using pronucleus injection techniques, the transgenic DNA fragment is injected to the zygote of the mouse. Then the injected zygote are transplanted into the fallopian tube of a pseudocyesis female mouse. It is then pregnant and give birth to its offsprings. After this, the tail-tip of the new born small mouse is cut and its gene group DNA is extracted to be identified by PCR to make sure that it carries the transferred gene fragment. The next step is to make the transgenic founder backcross with the wild-type C57BL/6J mouse. Then take the transgenic positive offsprings and confirm that the transferred gene is cardiac over expression by applying q PCR techniques and Western Blot-protein imprinted techniques. RNA taken from the cardiac of transgenic mouse is reverse transcribed into cDNA afterwards. Sequencing is done to determine the cardiac expression of mutant gene G100 S.(2) Research on cardiac hypertrophy phenotypes and the mechanisms of PRKAG2(G100S) transgenic mouse models: Define the wild-type littermate(WT) as a negative control group and the PRKAG2(G100S) transgenic mouse(TG) as an experimental group. Detect the cardiac of transgenic mouse and wild-type littermate by ultrasound and confirm whether cardiac hypertrophy exists among them. By weighing the cardiac dry weight and the body weight of the mouse, what can be known is that whether it is of statistical significance to confirm the ratio between cardiac weight and body weight. The histologic appearance of cardiac hypertrophy transgenic mouse can be acknowledged through cardiac HE staining and other histopathology. Then PAS staining is applied to identify intracellular accumulation of glycogen and AMPK kit detecting techniques are applied to show how the activities of mutant gene G100 S change in cardiomyocytes of transgenic mouse.(3) Research on arrhythmia phenotypes and the mechanisms of PRKAG2(G100S) transgenic mouse models: By using electrocardiogram examination, it can be sure whether arrhythmia phenotypes exist in transgenic mouse. Separate the cardiocytes of the adult transgenic mouse through confocal technique detections and confirm the homeostasis of calcium ions in the cells.Results:(1) Successfully built the vector sequence which contains this mutant gene. Successfully established PRKAG2(G100S) transgenic mouse models by using pronucleus injection techniques. By applying q PCR techniques and Western Blot techniques, it is verified that this mutant gene can both passage and over express stably in the cardiac of F2 transgenic mouse.(2)The ultrasound results showed that(WT vs TG):IVS-d(0.858±0.081 vs. 1.31±0.216 mm, n=6, p<0.01), IVS-s(1.211±0.184 vs. 1.695±0.144 mm, n=6, p<0.01), LVPW-d(0.687±0.114 vs. 1.063±0.159 mm, n=6, p<0.01), LVPW-S(1.013±0.108 vs. 1.370±0.083 mm, n=6, p<0.01) and LV Mass(94.11±7.25 vs. 193.23±54.41 g, n=6, p<0.01),which demonstrated the cardiac hypertrophy phenotypes existed in PRKAG2(G100S) transgenic mouse. HE staining proved its disorganized cardiocytes and there were necrotic vacuole and other histologic appearances. PAS staining showed there were large intracellular accumulation of glycogen in cardiocytes of the transgenic mouse. AMPK kit detecting techniques tested the cardiac tissues of wild-type and transgenic type respectively, the result was: n=3,P=0.0046. It showed statistical differences and proved that in comparing with the wild-type littermate, the cardiocytes activities of mutant gene G100 S in transgenic mouse decreased.(3) Temporarily the electrocardiogram monitor has not found arrhythmia of the mouse. But by using of confocal scanning, the result of number calculating among 138 S calcium waves showed WT vs TG(1.2±1.316 vs 6.3±2.312,n=10,P p<0.01), which confirmed that the mutant gene could cause the change of cardiocytes homeostasis.Conclusion: This research project successfully established the transgenic mouse models of PRKAG2(G100S) over expression. It demonstrates the G100 S mutant gene can cause cardiac hypertrophy of the transgenic mouse. The pathogenic mechanism are the same as the mutation pathogenic mechanism reported in CBS areas presently, which means cardiac hypertrophy can be finally induced by intracellular accumulation of glycogen through decreasing AMPK activities. Although the arrhythmia can not be detected by electrocardiogram examination, the homeostasis of intracellular calcium ions has changed by applying confocal techniques and rupture detection on separated cardiocytes of the transgenic mouse, which proved that arrhythmia phenotypes exist objectively. Just because the current electrocardiogram examination techniques can not discover it, other observation techniques can be considered at the late stage. Above all, the establishment of this transgenic mouse model demonstrates that the mutant gene can cause cardiac hypertrophy and provides good models for the further study of arrhythmia. It is of grate significance.