Patient-Specific Induced Pluripotent Stem-Cell Models For PRKAG2 Cardiac Syndrome

Background and Objectives:PRKAG2 gene mutation can lead to cardiac function disorder clinically,in which the typical features include ventricular pre-excitation,progressive conduction abnormality,cardiac hypertrophy,and excessive glycogen accumulation.The disease was defined as PRKAG2 cardiac syndrome and belonged to glycogen storage cardiomyopathy.A minority of patients with the syndrome presents with skeletal muscle dysfunction.Pathologic sections of the myocardial tissue from these patients revealed that there were absence of myocardial fibers disarray and fibrosis（or slight fibrosis in severe patients）,which is substantially different from hypertrophic cardiomyopathy.Although rare,PRKAG2 cardiac syndrome is an autosomal dominant inherited disease and occurs frequently in affected families.The diagnosis of the disease depends on genetic testing.More than 16 gene mutations in PRKAG2 causing amino acid replacements have been reported globally to date,consisting of 1 frameshift mutation and15 missense mutations;most of studies supported that these mutations changed the activity of adenosine monophosphate-activated protein kinase（AMPK）and lead to clinical manifestations.Characterization of the disease varies from different mutations;and even with the same mutation,affected members in one family have very different manifestations.As it is difficult to obtain cardiomyocyte samples of the patient with PRKAG2 cardiac syndrome,transgenic mice and knock-in mice were then used in previous researches.However,there has been debate about the change of AMPK activity affected by PRKAG2gene mutation.Moreover,the results were not convincing because of innate differences between human and animals.With the development of stem cell technology,somatic cells derived from patients can be reprogrammed and induced to pluripotent stem cells（iPSCs）,and then directed differentiated to cardiomyocytes,which is in favor of exploring the mechanism of a disease.Use of iPSCs can also overcome these issues such as differences among species and difficulty in cardiomyocyte sample collection for experimental researches.In addition,unlimited number of iPSCs can be provided for a study.In 2007,Jing Zhang et al reported a family characterized by familiar conduction abnormality,ventricular pre-excitation and cardiac hypertrophy.After genetic testing,they found that guanine in PRKAG2 gene exon 3（site 298 of cDNA）was replaced by adenine and then,glycine（amino acid site 100）was replaced by serine at the stage of translation.This mutation was thought to account for PRKAG2 cardiac syndrome.In following years,disease models from cell line of CCL13,zebrafish and mice were used to study the mechanism.However,these studies did not represent a real status of cardiomyocytes in the patients with PRKAG2 cardiac syndrome.To overcome the limitation,we make use of iPSCs technology and try to do deep investigation at the level of human.There is much less information about iPSCs model of PRKAG2 cardiac syndrome,and the exact mechanism of the disease has been unknown.In addition,there is absence of a study covering overall characteristics of cardiomyocytes,including glycogen content,AMPK activity,expression of AMPKγ2,cell size and cardiomyocyte function.We aimed to obtain cardiomyocytes from directed differentiation of induced pluripotent stem cells derived from patients with PRKAG2 cardiac syndrome and detect the characteristics of these cardiomyocytes.Methods:（1）Collect clinical data of the family members with PRKAG2 cardiac syndrome and perform genetic testing:we enrolled 9 persons,of which 6 were family members and 3 were partners of members.Health history,information of physical examination and objective workup were gathered.Additional investigations contained the level of myocardial enzyme,electrocardiogram（ECG）,echocardiography and dynamic ECG.Peripheral blood and/or urine were also collected for cell cultures of part 2.DNA of each candidate was extracted,exon 3 of PRKAG2 gene was amplified by polymerase chain reaction（PCR）and used to genetic testing.All candidates were grouped by results of genetic testing and clinical manifestations,and whole exome sequencing was performed in one member of each group.（2）iPSCs culture and directed cardiomyocytes differentiation:peripheral blood mononuclear cells（PBMCs）were isolated from peripheral blood for each candidate and urinary cells（UCs）were isolated from urine for most of candidates.Both of PBMCs and UCs were expanded according to protocols.After culturing enough cells,they were used to generate iPSCs using genome non-integrating episomal vectors（Epi5^TMM Episomal hiPSC Reprogramming Kit）.iPSCs were confirmed by different methods.Then,iPSCs were differentiated to cardiomyocytes（CMs）using the small-molecule-directed differentiation method.CMs were confirmed by immunostaining.（3）Detect characteristics of iPSC-CMs:iPSC-CMs were cultured for 30 to 90 days and then detected.Periodic acid-schiff（PAS）staining was used to qualitatively detect glycogen content.Immunocytochemistry was used to compute cell area of cardiomyocytes and then cell sizes were compared between groups.We only analyzed those cells with positive staining of cardiac specific protein troponin T（TNT）orα-actinin.Western blotting was used to detect protein level of AMPKα,p-AMPKα,AMPKγ2 in groups.AMPK activity was computed by the ratio of p-AMPKαand AMPKα.Whole-cell patch clamp technique was used to record cardiac action potentials（APs）and ionic currents.Results:（1）By whole exome sequencing analysis,two missense mutations causing amino acid replacements,R302Q and G100S,were found in PRKAG2 gene from the patientⅢ-3.In light of history and overall examination,these subjects were divided into three groups:patient group（PA group）,healthy kindred group（HK group）and healthy non-kindred group（HN group）.PA group included 4 family members,Ⅱ-1,Ⅲ-2,Ⅲ-3,andⅣ-2.Their common feature was that PRKAG2 gene mutation was validated by genetic testing.They presented with symptoms such as chest distress or palpitation except the patientⅣ-2;ventricular hypertrophy was found in echocardiography.Atrial fibrillation,bradycardia or bundle-branch block occurred in ECG.It was special for the patientⅣ-2.She was absence of symptoms,and the result of echocardiography was normal;whereas sinus bradycardia occurred in ECG and gene mutation was found by testing.HK group consisted of 2 family members,Ⅲ-1 andⅣ-3;genetic testing was normal in this group.They were absence of symptoms and had normal objective workup.HN group included 3partners（Ⅲ-1H,Ⅲ-2W,Ⅲ-3W）;genetic testing was normal.They were also absence of uncomfortable symptoms and abnormal workup;（2）We successfully reprogrammed somatic cells of 5 candidates into iPSCs,2（Ⅲ-2,Ⅲ-3）from PA group,1（Ⅳ-3）from HK group and 2（Ⅲ-2W,Ⅲ-3W）from HN group.Demonstrations of iPSCs were as follows:alkaline phosphatase detection was positive;immunocytochemistry revealed that iPSCs expressed pluripotency markers,including Oct-4,Sox-2,SSEA-4,TRA-1-60,TRA-1-81and Nanog protein.Real-time quantitative PCR showed that iPSCs expressed many pluripotency-associated genes,such as Nanog,REX1,ESG1,DPPA2,and UTF1.And,we successfully differentiate iPSCs into cardiomyocytes,which were confirmed by spontaneous contraction observed under microscopy and cardiac specific protein expression（TNI,TNT,andα-actinin）;（3）Characteristic detection of iPSC-CMs:By PAS staining,there was no significant difference in glycogen content between 3 groups,which showed that no glycogen accumulation occurred in iPSC-CMs derived from the patient.Cell area in PA group（514.1±288.2μm²）was significantly larger than that in HK group（325.0±289.2μm²,P<0.05）and HN group（283.6±233.4μm²,P<0.05）,which demonstrated that iPSC-CMs derived from the patient presented with enlargement characteristic.We found that AMPK activity in PA group（0.94624±0.06390）remained similar to that in HN group（0.85370±0.03645,P=0.08）,and both of them were significantly higher than that in HK group（0.73588±0.12625,P<0.05）.It suggested that there was no change of AMPK activity in iPSC-CMs derived from the patient.AMPKγ2level in PA group was significantly higher than those in HK group and HN group,which indicated that gene mutation increased the level of encoded protein.Using Whole-cell patch clamp,we recorded ventricular-or atrial-like cardiac APs in PA group and HK group,but not in HN group.We observed high level of sodium(I_Na)and calcium ionic current(I_Ca)in PA group（1500-2000pA）,which did not happen in HK group.Conclusions:This study successfully differentiated iPSC-CMs derived from patients with PRKAG2 cardiac syndrome;the patient-derived iPSC-CMs were demonstrated to show some characteristics of the disease,such as cell enlargement,increase of AMPKγ2,and high level of I_Naa and I_Ca;whereas,these cells were absence of glycogen accumulation and evident change in AMPK activity.The number of iPSC-CMs that we analyzed was yet too less to extensively evaluate their character.It is needed for further research to improve culture conditions and produce sufficient and relatively mature cardiomyocytes.In conclusion,these iPSC-CMs we established can be a useful model for further trials to explore the pathogenesis of PRKAG2 cardiac syndrome.