Identification And Functional Analysis Of A PRKAG2 K291R Variant Associated With PRKAG2 Cardiac Syndrome

PRKAG2 genetic variation can lead to PRKAG2 cardiac syndrome,which is characterized by ventricular preexcitation,myocardial hypertrophy and glycogen accumulation.In this study,we screened a PRKAG2 gene variant in patients with PRKAG2 cardiac syndrome,and explored the possible pathogenic mechanism of the new PRKAG2 gene variant.The main research contents and results are as follows:1.Two new genetic variants of PRKAG2 gene,Q172 L and K291,were identified in the same patient.EGFP labeled PRKAG2-WT and PRKAG2-Q172 L,PRKAG2-K291 R,PRKAG2-Double(double variant)plasmids were transfected into HEK293 cells,then Western Blot was used to detect changes in PRKAG2 protein expression and AMPK activity.To the Q172 L variant,neither PRKAG2 protein expression nor AMPK activity were changed,however,PRKAG2 protein expression and AMPK activity decreased significantly in the K291 R and Double variant.The immunofluorescence analysis showed that the Q172 L,K291R and Double variatit did not affect the localization of PRKAG2 protein.All these results suggested that the K291 R variant had obvious pathological effect.2.Wild type and K291 R variant plasmids were transfected into H9C2 cardiomyocytes,these results of Western Blot were consistent with the results from HEK293 cells and confirmed that the K291 R variant affected the expression of PRKAG2 protein but not its localization.The protein degradation pathway was further studied.The protein expression of PRKAG2 was detected by Western Blot after treatment with proteasome inhibitor(MG132)and lysosome inhibitor(CHL).The results showed that compared with CHL treatment,the protein expression of K291 R variant treated with MG132 increased more significantly,indicating the reduced stability of K291 R variant through the protease pathway.3.Wild type and K291 R variant plasmids were transfected into H9C2 cardiomyocytes,compared with the WT,the K291 R variant did not affect the AKT/m TOR,e EF2kinase/e EF2,Calcineurin A/NFATC3,NF-κB and STAT3 pathway,but led to inactivation of the FOXO3 pathway.The result further confirmed the decrease in Mu RF1 protein expression by Real-time quantitative PCR and Western Blot.And then the expression of proteins associated with endoplasmic reticulum stress(s XBP1,p-e IF-2a,ATF4 and ATF6),autophagy(Beclin,P62,LC3-I and LC3-II),and fibrosis(SMAD2 and SMAD3)were analyzed by Western Blot.These results showed that the K291 R variant did not induce endoplasmic reticulum stress,autophagy or altered fibrosis pathways.4.Wild type and K291 R variant plasmids were microinjected into zebrafish embryos,then we detected the cardiac function indexes,performed HE staining and glycogen staining.These results showed that the K291 R variant affected the normal cardiac function of zebrafish,resulting in the increase of heart rate(HR),the decrease of fractional shortening(FS),the thickening of ventricular wall and the accumulation of cardiac glycogen.Main conclusions:1.PRKAG2 K291 R is a genetic variant associated with PRKAG2 cardiac syndrome.K291 R variant can reduce PRKAG2 protein expression and AMPK activity2.PRKAG2 K291 R variant affects the FOXO pathway related to cardiac hypertrophy.By inhibiting the transcriptional activity of FOXO-3a,FOXO loses its effect on its downstream target gene Mu RF1 and inhibits protein hydrolysis,resulting in cardiac hypertrophy.