The Effect Of Protein Kinase C Isozymes On HERG Potassium Channel Expression

Arrhythmias, especially severe ventricular arrhythmias are leading causeof deaths for cardiovascular diseases. Therefore, cardiovascular research hasbeen focused on revealing the molecular mechanism of arrhythmia.Abnormality of struscture and/or dysfunction o fsarcolemmal ion channelsresulting either congenital genetic deficiency or acquired factors primarilycontribute to arrhythmias.Potassium channels constitute a highly diverse class of ion channel.Among them, the rapidly activating delayed rectifier K current (I_Kr) has animportant influence on maintaining myocardial action potential plateau phaseand phase3repolarization phase. The pore-forming subunit of I_Kris encodedby hERG gene （human ether-a-go-go-related gene）. Congenital hERG genemutations cause cardiac channelopathies. Cardiovascular diseases （such ashypertension myocardial ischemia） are associated with abnormal function ofhERG/I_Krpotassium channels and the down-regulation of hERG function ledsto acquired LQT syndrome. LQT syndrome is often associated with a varietyof ventricular arrhythmias （especially may cause torsades de pointes and fatalarrhythmias）, with high risk of sudden cardiac death （SCD）. Therefore, it is ofsignificanct importance to study on pathological remodeling of hERG/I_Krpotassium channels function.Protein kinase C （PKC） is a group of Ca²⁺phospholipid-dependentserine-threonine protein kinase. So far, at least ten kinds of PKC isozymeshave been identified. These isozymes are classified based on their structureand activation requirements into three subgroups: conventional PKCs （α、βI、βII、γ）；novel PKCs （δ、ε、π、θ）；and atypical PKCs （ζ、/λ）. Studies havedemonstrated that PKC is an important mediator of humoral factors andneurotransmitter in the regulation of hERG/I_Krpotassium channels function. Increased neurohumoral factors in pathological conditions change hERG/I_Krpotassium channels function through the activation of PKC.Studies have proved that stimulation of α_1Areceptor down regulateshERG/I_Krcurrent through the PKC and PKA pathways in isolated guinea pigventricular cells and Xenopus oocytes transfected with hERG cDNA. ThePKC pathway also mediates the down-regulation of hERG/I_Krcurrent by M₃cholinergic receptor. Our previous studies have shown that Ang II exerts acuteinhibition on hERG/I_Krcurrent in isolated guinea pig ventricular myocytes andheterologous expression system. Direct PKC activation by PMA or OAGproduces an inhibitory effect on hERG/I_Krcurrent. These experiments indicatethat the activation of PKC play acute inhibitory effect on hERG/I_Krchannel.However, studies have proved that chonic exposure to PMA or α_1Areceptoragonist PE for24h, increases hERG channel protein abundance in stablyexpressing hERG-HEK293cells.It is known that different PKC isozymes have different downstreamtarget molecules to exert specific functions. In artery smooth muscle cells,endothelin-1inhibits IKcurrent through PKCα, but Ang II inhibits IKcurrentvia PKCε. Activation of PKCβII or PKCε could up-regulate the expressionof I_Kscurrent, but α、βI and δ isozymes has no significant effect on channelfunction. So far, whether different PKC isozymes could specifically regulatehERG/I_Krchannel is unclear.Therefore, the present study was designed to investigate the effect ofdifferent PKC isozymes （mainly for the cPKCs and PKCε isozymes） on hERGchannel abundance in heterologous expression systems by using molecularbiological methods. The result will be of important significance for theunderstanding mechanism of electrophysiological remodeling in pathologicalconditions and thus find novel target for treating arrhythmis.Objective:To observe the chronic regulation of the hERG channels proteinexpression by cPKCs and PKCε isozymes. Methods:Stably expressing hERG-HEK293cells were incubated withnon-selective or selective PKC isozymes activators or activator peptide for24h. The total proteins were extracted by SDS-PAGE, then transferred to NCmembranes, and blocked with5%fat-free milk for2-4h. The membranes wereincubated with primary antibody against hERG and then with fluorescentlylabeled secondary antibodies.The anti-GAPDH antibody was used as internalloading control. Quantification of the signals was performed by usingOdyssey9120Infrared Imaging System, and analysis of the band density wasdone with the software. The band intensities were normalized by GAPDH andsummary data for hERG protein abundance were presented as a percentage ofthe control group. All of data are presented as means±SEM. The dataanalysis was performed with Origin software （version Pro7.0）. The nrepresents the repetitive times. The statistical significance of the differencesbetween groups was evaluated using Student’s unpaired t test and differenceswith P <0.05were considered statistically significant.Results:（1） Effects of non-selective PKC isozymes activators on hERGpotassium channels protein expression: Western blot analysis showed thatincubation of PMA （100nM） for24h increased mature （155kD） andimmature （135kD） protein expression to140.8±3.0%（P<0.001, n=3） and143.2±5.0%（P<0.001, n=3）, respectively. OAG （10μM） also enhanced themature and immature protein expression to148.0±6.0%（P<0.001, n=3） and126.5±3.8%（P<0.001, n=3）, respectively. Compared with PMA, OAG hadsimilar effect on the mature protein （P>0.05）. For the immature protein, theaction of OAG was weaker than PMA （P<0.05）. The results showed that thechronic incubation of non-selective PKC isozymes activators could markedlyincrease the expression of the hERG potassium channel protein （both matureand immature forms）.（2） Effects of selective PKC isozymes activators on hERG potassiumchannels protein expression:Firstly, Western blot analysis showed that incubation of selective cPKCs isozymes activators Ingenol （200nM） for24h increased the mature andimmature protein expression to143.0±7.3%（P<0.01, n=3） and145.1±6.9%（P<0.01, n=3）, respectively. Next, selective PKCε isozyme activatorsFR236924（200nM） also enhanced the mature and immature proteinexpression to129.7±3.6%（P<0.001, n=4） and115.8±0.8%（P<0.001, n=4）,respectively. Compared with Ingenol, FR236924had similar effect on themature protein （P>0.05）. For the immature protein, the action of FR236924was weaker than Ingenol （P<0.01）. The results showed that both cPKCs andPKCε up-regulate the expression of the hERG potassium channel, and the roleof cPKCs was more potent than PKCε.Combined application of Ingenol （200nM） and FR236924（200nM） for24h increased the mature and immature protein expression to153.8±5.7%（P<0.001, n=3） and158.9±5.4%（P<0.001, n=3）, respectively. Comparedwith Ingenol alone, combination group had similar effect on both mature andimmature protein （both P>0.05）. Compared with FR236924alone, the actionof combination group was more potent than FR236924on both mature andimmature protein expression （P<0.01or P<0.001）. The results hint thatcPKCs and PKCε may have the same downstream target molecular mediatingthe chronic regulation of hERG potassium channel.（3） Effects of selective PKC isozymes activator peptide on hERGpotassium channels protein expression:The hERG-HEK293cells were respectively incubated with perforatingagent Saponin （200nM） alone, Saponin （200nM） plus selective cPKCsisozymes activator peptide （200nM） or Saponin （200nM） plus selectivePKCε isozyme activator peptide （200nM） for24h. Western blot analysisshowed that Saponin alone had no effect on both mature and immature formsof hERG protein （both P>0.05）. Wherease, cPKCs activator peptide （200nM）increased the mature and immature protein expression to138.7±4.5%（P<0.01, n=3） and120.2±3.2%（P<0.01, n=3）, respectively. PKCε activatorpeptide （200nM） also enhanced the mature and immature protein expressionto127.2±0.4%（P<0.01, n=3） and116.6±1.6%（P<0.01, n=3）, respectively. Compared with cPKCs activator peptide, the action of PKCε activator peptidewas weaker than cPKCs activator peptide on the mature protein expression（P<0.05）. The results showed that both cPKCs and PKCε up-regulate theexpression of the hERG potassium channel, and the action of cPKCs wasmore potent than PKCε. The results are consistent those of selective PKCisozymes activators.Next, the combined ation of selective cPKCs and PKCε isozymesactivator peptide was observed in the presence of perforating agent Saponin（200nM）. Western blot analysis showed that Saponin had no effect on bothmature and immature protein （both P>0.05）. Combination of two isozymesactivator peptides enhanced the mature and immature protein expression to139.3±1.8%（P<0.001, n=3） and124.5±2.4%（P<0.001, n=3）, respectively.Compared with cPKCs activator peptide alone, combination group had similareffect on both mature and immature protein （both P>0.05）. Compared withPKCε activator peptide alone, the action of combination group was morepotent than PKCε activator peptide on both mature and immature proteinexpression （P<0.01or P<0.05）. The results indicated that cPKCs and PKCεmay have the same downstream target molecular mediating the chronicregulation of hERG potassium channel. The results are consistent withcombined selective PKC isozymes activators.Conclusion:（1） In heterologous expression systems, the chronic activation of PKCmarkedly increases the hERG channel expression （including mature andimmature protein）.（2） Both cPKCs and PKCε up-regulate the expression of the hERGpotassium channel, and the role of cPKCs was more potent than PKCε.