The Mechanism Of PP2A Regulatory Subunits Targeting Dephosphorylated Myocardial Cav1.2 Calcium Channels

BackgroundCav1.2 Ca2+channels are one of important Ca2+ handling protein in cardiac excitation-contraction(E-C)coupling.Reversible phosphorylation of Cav1.2 Ca2+channelscoordinately controled by protein kinase A(PKA)and protein phosphatase 2A(PP2A)plays an important role in maintaining efficiency of cardiac EC coupling.Although the PKA-regulated phosphorylation of Cav1.2 has been extensively studied,it is largely unknown the precise mechanisms that PP2A counteracts increase in Cav1.2 channel activity by PKA,especially the B subunits-mediated activation of PP2A holoenzyme and site-specific Cav1.2 dephosphorylation.Objective1.Under the physiological condition,the basal phosphorylation state of Cav1.2 phosphorylation is largely determined by the activity of protein phosphotases,and PP2A is the major phosphotase involved in regulating the phosphorylation of Cav1.2.However,it is not clear which isoforms of the PP2A regulatory subunits account for PP2A-induced Cav1.2 dephosporylation process and the mechanisms involved in this precess.Thus,first of all,we aim to identify the regularoty subunits of PP2A involved in Cav1.2 signaling complex under physiological condition.2.Under the pathological condition of heart failure,changes in Cav1.2 calcium channel activity resulted from the decreased Cav1.2 phosphorylation lead to a decrease in cardiac E-C coupling efficiency and consequent impaired contraction.Parallely,significant enhanced expressions of PP2A regulatory subunits are evident in ischemic or non-ischemic heart failure(HF).But it is not known whether any link exists between the decreased Cav1.2 phosphorylation and increased PP2A regulatory subunits expression,and the mechanisms underlying the phosphorylation level of Cav1.2 mediated by PP2A regulation subunits.Therefor,we aim to explore the regularoty subunits of PP2A account for dephosphorylation of Cav1.2 under the pathophysiological conditions and the underlying mechanisms.To answer the questions mentioned above,we conducted experiments as follows:Part 1 The PP2A regulatory subunit isoforms and mechanisms involved in dephosphorylating at Ser1928-Cav1.2 under the physiological conditionMaterial and methods1.IP assay was performed to identify the regulatory subunits of PP2A involved in the Cavl.2 dephosphorylation process.2.PP2A B' regulatory subunits were overexpressed in H9c2 cells to investigate the function of B' regulatory subunits on Cav1.2 dephosphorylation.3.Dephosphorylation assay was used to verify the effect of PP2A regulatory subunits on dephosphorylating at Ser1928-Cav1.2 in vitro.Results1.IP assay showed that regulatory subunits B' family but not B/B”families was capable of interacting with Cav1.2 signaling complex in mouse ventricle myocytes.Among the PP2A B' family,only PPP2R5A,PPP2R5D and PPP2R5E existed in the p-Ser1928-Cav1.2 signaling complex.2.Both total protein expression of Cav1.2 and phosphorylated Cav1.2(p-Ser1928-Cav 1.2)were increased,while the phosphorylation level of Ser1928-Cav1.2 was reduced in response to overexpression of different subunits of B'family in H9c2 cells,respectively.In addition,overexpression of PPP2R5C and PPP2R5D caused a significant increase in the mRNA expression of Cav1.2.3.The increased protein and mRNA expression of Cav1.2 and p-Ser1928-Cav1.2 induced by the overexpressed B' family was abolished by the treatment of okadaic acid(OA)at a concentration of 3 nM,which selectively inhibits PP2A.But the decreased phosphorylation level of Cav1.2 did not be reversed by OA treatment.4.The increased Cav1.2 and p-Ser1928-Cav1.2 protein expression induced by the overexpressed B' family were returned to the basal level by blocking either the protein synthesis(CHX)or degradation pathway(Lis),respectively,suggesting the B'family involved in either protein synthesis or degradation of Cav1.2.However,B'sunbunits overexpression-induced decreased phosphorylation level of Cav1.2 was not changed under the condition of CHX or Lis treatment.5.In vitro assay showed that incubation of purified B' subunits with purified p-Ser1928-Cav1.2 respectively,only PPP2R5A,PPP2R5D and PPP2R5E were able to directly dephosphorylate Cav1.2.SummaryUnder the physiologic condition,PP2A regulatory subunits PPP2R5A,PPP2R5D and PPP2R5E dephosphorylated at Ser1928 of Cav1.2,via dephosphorylating at the Ser1928 site of Cav1.2 directly or affecting the protein synthesis or the protein stability.Part 2 The PP2A regulatory subunit isoforms involved in dephosphorylating at Ser1928-Cav1.2 under the path physiological conditionMaterial and methods1.Isolated neonatal rat ventricle myocytes(NRVMs)were treated with isoproterenol(ISO)to prepare the hypertrophic model in vitro.2.Eight-month-old male C57BL/6 mice were subcutaneous injection of ISO(5 mg/kg/d for 7 days)to prepare the HF mouse models.3.Mutants of B' subunits at different sites of PKA phosphorylation and siRNAs targeting to B' subunits were construced,repectively,to verify the specific function of PP2A regulatory subunits on p-Ser1928-Cav1.2.Results1.In cultured NRVMs treated with ISO,both the phosphorylation of Ser1928-Cav1.2 and the protein expression of PP2A regulatory subunits B' family were time-dependently elevated,reaching their peak 4 h after and then declined with the extension of the ISO processing time.Furthermore,the amount of PPP2R5A,PPP2R5D and PPP2R5E combined with p-Ser1928-Cavl.2 was peaked at 4 h after ISO treatment and declined later.2.In the failing hearts,the protein expression of both total Cav1.2 and p-Ser1928-Cav1.2 were enhanced,but the phosphorylation level of Cav1.2 was reduced.In addition,the scaffording subunits,catalytic subunits and the regulatory subunits B' family of PP2A were all upregulated,besides the regulatory subunits B and B" family.3.By IP assay with anti-p-Ser1928-Cav1.2 antibody,the binding of PPP2R5A,PPP2R5D and PPP2R5D to p-Ser1928-Cav1.2 was enhanced in the samples from failing hearts.Furtheremore,by IP assay with anti-PP2A catalytic subunits antibody,the binding between PPP2R5D and PP2A catalytic subunit was increased,in contrast,other regulatory subunits of B' family were dissociated from the activated PP2A holoenzyme,indicating an important role of PPP2R5D in mediating PP2A-induced Cav1.2 dephosphorylation.4.H9c2 cells overexpressed with different regulatory subunits of B' family were treated with ISO to activated PKA.Western blot analysis showed that,as compare to empty vector,the phospholation level of Ser1928-Cav1.2 was increased by PPP2R5A,PPP2R5B and PPP2R5C while that was decreased by PPP2R5D and PPP2R5E,implying that the activity of PP2A holoenzyme was determined by the phosphorylation of regulatory subunits B' family themselves,as a result,affected phosphorylation level of Ser1928-Cav1.2.5.The effects of PKA-dependent PPP2R5D phosphorylation on Ser1928-Cav1.2 were further confirmed in H9c2 cells transfected with wild-type or mutated PPP2R5D repectively.In the presence of PKA,the ability to dephosphorylate Cav1.2 was diminished by the mutation in Ser566A of PPP2R5D to abolish the activity of PP2A holoenzyme.While,the ability of PP2A-induced dephosphorylation of Cav1.2 was not affected by the mutants including Ser53A,Ser68A and Ser81A of PPP2R5D in comparison with WT.6.Knockdown the expression of PPP2R5D by siRNAs in NRVMs,the protein expression of Cav1.2 was reduced and the phosphorylation of Ser1928-Cav1.2 was increased.However,no compensation of other B' regulatory subunits was documented.SummeryUnder the pathphysiological condition,the increased PPP2R5D accounted for an enhancement of PP2A holoenzyme activity and the consequence of decreasing phosphorylation at Ser1928-Cav1.2.