PP2A Knockout In Adult Mouse Heart Causes Mouse Heart Failure

Protein phosphorylation is a major control mechanism of a wide range of physiological processes and plays an important role in cardiac pathophysiology.Serine/threonine protein phosphatases(PP1,PP2A,PP2B and PP2C)control the dephosphorylation of a variety of cardiac proteins,thereby fine-tuning cardiac electrophysiology and function.Specificity of protein phosphatases type-2A is achieved by multiprotein complexes that target the catalytic subunits to specific subcellular domains.PP2A is a heterotrimer consisting of an A structural subunit,a B regulatory and a C catalytic subunit,and the B subunit gives the complex substrate specificity.Four families of B subunits(PR55 or B,PR61 or B’,PR72/130 or B"and PR93/PR110 or B"’)exist,each containing multiple members.There are two isoforms α and β of A and C subunits in eukaryotes.The regulatory B subunits play key roles in controlling PP2A substrate specificity,cellular localization,and enzymatic activity.Each family of B subunits contains several isoforms that can bind the AC dimer in a mutually exclusive manner.The a form of PP2A catalytic subunit was encoded by mouse gene ppp2ca.Conventional knockout of ppp2ca allele leads to embryonic lethality in mice.To investigat PP2A in adult mice cardiac function,we set up ppp2ca conditional knockout mice models by Myh6-MerCreMer tool mice with ppp2canflox/flox mice.Total RNA were isolated from mice’s hearts and the mutant form of PP2ACαknockout mRNA in these mice was verified by RT-PCR,which was 200 bp shorter than control wild type,PP2ACα gene level was decreased in knockout mice by quantitative real-time PCR examine.To examine PP2ACa at the protein level,western blot was performed to detect the catalytic subunits of PP2A.Protein levels of PP2AC decrease significantly in knockout mice.Phosphatase activity was found elevated in knockout in comparison to control controls.Cardiac function of mice were determined by echocardiography.Cardiac function of knockout mice were the same after one day tamoxifen injection tamoxifen injection.Cardiac specific knockout mice as well as wild control mice at 3 month,were treated with tamoxifen by intraperitoneal injection once a day for 3 days at a dosage of 20 mg/kg per day.PP2A activity decreased leads to increased cardiomyocyte hypertrophy since 10 days after tamoxifen injection and fibrosis at 60 days after tamoxifen injection.Cardiac hypertrophy is frequently associated with gene re-expression during fetal and perinatal development,and with the upregulation of some cardiac proteins,such as ANP(atrial natriuretic peptide),BNP(brain natriuretic peptide),α-MHC(α-myosin heavy chain)and β-MHC(β-myosin heavy chain).Quantitative real-time PCR of myocardial cell RNA showed that these genes were significantly upregulated in the knockout mice hearts compared with controls at 60 days after injection.Hypertrophy markers were highly expressed in knockout heart.The results showed in knockout mice heart,B55α,B56ε express increased,while the expression of B55β decreased with significant difference compared with the control group.B55β,B56α,B56β,B56γ,B56γ2,B56ζand B",also express higher.While we found no differences regarding the total amount of Akt between the different groups,there was a significant decrease of Akt-phosphorylation in knockout mice.GSK-3β,the β-catenin negative regulator,is activated both directly and through AKT inhibition by PP2A.In knockout mice,phosphorylated GSK3β was detected decreased.Controversially,we found significantly lower concentrations of β-catenin in PP2A knockout mice with an increase of β-catenin phosphorylated on Ser552.In summary,our study proved the key function of PP2A in adult mice cardiac function.By phenotyping the myocardial cell specific ppp2ca null mice,we found knockout mice became cardiac hypertrophy,fibrosis,and finally heart failure,and the regulation of the Akt/GSK3β/β-catenin pathway is severely disturbed in PP2A knockout mice.