Pathogenesis Of PRKAG2 Cardiac Syndrome Caused By A Novel Mutation PRKAG2 G100S In Chinese

Background:PRKAG2 G100S which was identified by Doctor Jing Zhang in 2007 is a novel PRKAG2 gene missense mutation and it was responsible for familial cardiac hypertrophy with conduction abnormity and ventricular preexcitation of Chinese. The patients presented with cardiac hypertrophy (56%), preexcitation syndrome (46%), sinus node dysfunction, advanced atrioventricular block, even sudden death. Atrial arrhythmia, such as atrial fibrillation and atrial flutter also coud been observed in some family members. The clinical phenotype in this Chinese family with PRKAG2 cardiac syndrome corresponded to general characters of the foreign families. After direct DNA sequencing was used, glycolamine (100) to serine, in exon 3 of PRKAG2 was identified and shown to be present in the living affected family members. The mutation results from guanine (G) substituted for adenine (A) at nucleotide 298. Therefore, we believe that PRKAG2 G100S could be the cause of PRKAG2 cardiac syndrome in Chinese people. In 2001, Gollob et al proposed hypothesis that PRKAG2 cardiac syndrome could be a novel cardiac glycogenosis syndrome, and they believed that genetic defects in PRKAG2 may lead to a phenotype of hypertrophic cardiomyopathy with ventricular pre-excitation by virtue of the massive accumulation of glycogen. Some studies suggest that PRKAG2 gene may be involved in the heart of the development process and lead to the formation of the heart bypass, so the identification of the PRKAG2 genetic defect should lead to an understanding of the pathogenesis of accessory atrioventricular connections in relate to the developing heart and of the pathogenesis of cardiac hypertrophy in relate to imbalance of energy metabolism. However, recent studies found that pathogenesis of cardiac hypertrophy could not be fully ascribed to accumulation of glycogen in myocardial cells. Abnormal activation of AMPK could lead to cardiac hypertrophy by affecting intracellular signal transduction pathways. PRKAG2 G100S is a novel mutation which was identified the first time, so pathogenesis of PRKAG2 cardiac syndrome in Chinese people is worth studying. To understand the effect of PRKAG2 G100S mutation on glucose metabolism and cardiac myocyte hypertrophy marker gene expression, we over-expressed PRKAG2 G100S in neonatal rat cardiomyocytes and H9c2 cells in order to define whether PRKAG2 G100S would result in diversification in intracellular ANP, a-MHC,β-MHC gene level and glycogen content and to clatify the pathogenesis of PRKAG2 cardiac syndrome in Chinese people.Objective:To investigated the effect of PRKAG2 G100S mutation on glucose metabolism in myocardial cells, Ca2+homeostasis and cardiac hypertrophy marker gene level such as ANP,α-MHC,β-MHC. Methods:(1) To construct recombinant adenovirus vector carrying wide type and mutants of human PRKAG2 gene:PRKAG2 G100S-IRES-EGFP and PRKAG2-IRES-EGFP were acquired by the overlapping PCR, and were cloned directly into entry vector pDONR221 by using Invitrogen's GatewayTM technology. Then, along with the BP and LR recombination reactions finished, the recombinant adenovirus vector containing human PRKAG2 G100S gene is constructed. The sequencing sequence was right. The pAd-PRKAG2 G100S-IRES-EGFP and pAd-PRKAG2-IRES-EGFP were digested by Pac I, packaging, amplification and purified. PCR technique was applied to detect the target gene. The titre of Ad-PRKAG2-EGFP and Ad-PRKAG2 G100S-EGFP was measured with the aid of enhanced green fluorescence protein (EGFP) expression. (2) Neonatal rat cardiomyocytes was cultured and was infected with recombinant adenovirus vector carrying wide type and mutants of human PRKAG2 gene:Neonatal rat cardiomyocytes was cultured and was infected with recombinant adenovirus vector carrying wide type and mutants of human PRKAG2 gene. Western Blot technique was applied to verify the expression of PRKAG2 protein. (3) Determine of intracellular free Ca2+:H9c2 cells was cultured and was infected with recombinant adenovirus vector carrying wide type and mutants of human PRKAG2 gene. It was determined after the cells incubated with Rohd-2/AM that Rohd-2 ratio in H9C2 cells before and after transfected 48 hours with Ad-EGFP, Ad-PRKAG2 and Ad-PRKAG2 G100S. (4)Determine of intracellular ANP,α-MHC,β-MHC gene level and glycogen content:Neonatal rat cardiomyocytes was cultured and was infected with recombinant adenovirus vector. Intracellular ANP,α-MHC,β-MHC gene level were determined by real-time qPCR technique. At last, glycogen contents in neonatal rat cardiomyocytes were detected by PAS.Results:(1) Restriction enzyme digestion analysis and the sequence analysis confirmed that PRKAG2 G100S gene was successfully inserted into the adenovirus vector. Myocardial cells which were transfected with Ad-PRKAG2 G100S-EGFP gave off strikingly bright green fluorescence, which implicated PRKAG2 protein was over-expressed. The titre of purified recombinant adenovirus Ad-PRKAG2-EGFP was 7.8×108 ifu/ml and the titre of Ad-PRKAG2 G100S-IRES-EGFP was 8.4×108ifu/ml. (2) Neonatal rat cardiomyocytes were successfully cultured and normal or mutant PRKAG2 gene was over-expressed in neonatal rat cardiac cells. Green fluorescence was seen in the cells infected with different recombinant adenovirus vectors. PRKAG2 protein was verified to be expressed successfully in neonatal rat cardiac myocytes by Western Blot. (3)Normal or mutant PRKAG2 gene was over-expressed in H9c2 cells and PRKAG2 protein was verified to be expressed successfully by Western Blot. Intracellular free Ca2+ assay using Rohd-2/AM showed that intracellular free Ca2+concentration ([Ca2+]i) in H9c2 cells which were transfected with recombinant adenovirus vectors in 24-48 hours in mutation (GS) group (12.72±7.68) was no significant difference with wild-type(PK)group (17.28±7.79) and GFP groups (19.33±5.50) (P> 0.05). In 48-72 hours, intracellular free Ca2+concentration ([Ca2+]i) in H9c2 cells which were transfected with recombinant adenovirus vectors in mutation (GS) group (24.18±2.43) was no significant difference with wild-type(PK)group (26.24±4.77) and GFP groups (26.04±5.71) (P> 0.05). (4) When neonatal rat cardiomyocytes was infected with recombinant adenovirus vectors in 48-72 hours, ANP mRNA level in mutation (GS) group (1.00±0.04) was no significant difference with wild-type(PK)group (1.00±0.10) and blank control (GFP) group (1.00±0.06)(P>0.05). In addition,α-MHC mRNA level in mutation (GS) group (1.00±0.13) was no significant difference with wild-type (PK) group (1.00±0.00) and GFP groups (1.05±0.38) (P>0.05). As toβ-MHC mRNA level, no significant difference was also found in GS group (1.04±0.33)and wild-type(PK) group (1.00±0.04) and blank control (GFP) group (1.09±0.53) (P>0.05). PAS using dyeing kit revealed that more glycogen was diffusely distributed in the cytoplasm in mutant group, however, less glycogen in WT and blank control group.Conclusions:(1) The recombinant adenovirus containing human PRKAG2 G100S gene is successfully constructed and also expressed in myocardial cells. It will be helpful for the further study on PRKAG2 gene mutation. (2)PRKAG2 G100S mutation resulted in intracellular glycogen accumulation, but it failed to result in significant change in intracellular Ca2+, ANP, andα-MHC,β-MHC mRNA level. This implied that accumulation of intracellular glycogen is probably the key of pathogenesis of PRKAG2 cardiac syndrome in Chinese people. Furthermore, inappropriate activation of AMPK secondary to the G100S PRKAG2 mutation couldn't be associated with imbalance of calcium homeostasis. (3)PRKAG2 G100S could mainly result in disorder of myocardial cellular energy metabolism, and it had probably few disturbances in ANP,α-MHC andβ-MHC mRNA level.