The Mechanisms Underlying MG53 Regulation Of The Expression Of Cardiac Auxilliary Subunit KChIP2 And The Role Of Which In Cardiac Electrical Stability

A tripartite motif (TRIM72) family member protein named MG53 was identified in 2009. Mitsugumin 53 (MG53), has a molecular weight of 53 kD, and contains the prototypical tripartite motif, which includes a RING, B-box and coiled-coil moiety at the amino terminus and a SPRY motif at the carboxyl terminus. MG53, a muscle-specific protein, is specifically expressed in skeletal and cardiac muscle and predominantly localized on intracellular vesicles, defined its role in mediating the dynamic process of membrane fusion and exocytosis in striated muscle. Many studies demonstrate that MG53 is closely related to the repair of the injured plasma membrane. Once plasma membrane is injured, MG53 which interacting with caveolin-3 will recruite MG53-containing vesicle to the injury sites and bud from the cell membrane for membrane-patch formation by sensing the extracellular oxidative environment.Myocardial cell membrane of transient outward potassium channel(transient outward potassium channel, Ito) is one of the voltage-dependent potassium channels, membrane potential is activated during depolarizing with properties of rapid activation and inactivation, it is a major K+ current to form action potential (action potential, AP) repolarization of 1 stage, plays an important role in changing AP duration and the regulation Ca2+. A variety of pathological conditions such as myocardial hypertrophy, myocardial infarction, diabetes, etc. can cause extended APD, QT interphase discrete, leading to ventricular arrhythmia and even sudden cardiac death, confirmed the mechanism associated with the down rugulation of Ito. KChIP2 is the obligatory beta subunit of cardiac transient outward current (Ito), which is essential for regulating Ito function. KChIP2 protein levels increase with the development of heart, which determines the developmental increase of cardiac Ito. Furthermore, the transmural gradients of KChIP2 transcription across the free ventricular wall (epicardial myocytes>mid-cardial myocytes>endocardial myocytes) in many species contribute to the Ito transmural grandients which are critical determinants of myocardial function. In multiple heart diseases, such as hypertrophy, infarct cardiomyopathy, heart failure, etc. Ito amplitudes are consistently decreased, predisposing the heart to the risk of ventricular arrhythmia. A series of studies demonstrate that decrease in KChIP2 protein expression is an important mechanism for the downregulation of Ito in diseased hearts. Therefore, KChIP2 plays an important role in maintaining cardiac electrical stability. Unfortunately, the mechanisms underlying physiological and pathological regulation of KChIP2 expression and Ito function are largely unknown.Reduced Kv2.1-mediated potassium currents, the anomalies of AP and arrhythmia have been found in MG53 knockout mice. It suggested that, MG53, a heart specifically expressed gene, played an important role in the ion channel and cardiac electrical stability. But that’s the only one finding reported MG53 on cardiac ion channels and cardiac electrical stability, MG53 on other function and mechanism of ion channels has not yet been in-depth study.Our preliminary data demonstrate that adenoviral overexpression of MG53 increased KChIP2 protein levels in cardiomyocytes, whereas MG53 knockdown by RNA interference decreased KChIP2 protein expression. Concomitantly, MG53 overexpression in cardiomyocytes increased the amplitude of Ito, while MG53 knockdown decreased cardiac Ito. These findings implicate that MG53 is an intrinsic regulator of KChIP2 expression in the heart.Based on the findings, we intend to address the following important questions in this project. First, the role of MG53 in the regulation of KChIP2 expression and Ito function in neonatal rat ventricular cardiomyocyte (NRVM) and adult rat ventricular cardiomyocyte (ARVM). Second, the regulation of MG53 in calcium signaling in adult rat ventricular cardiomyocyte Third, what are the molecular mechanisms underlying MG53 regulation of KChIP2 expression in cardiomyocytes. Through these efforts, we hope explore novel mechanisms participating in maintaining cardiac stability and new therapeutic targets for the treatment of arrhythmia in heart diseases.Methods:In NRVM and ARVM, the cells were infected with recombinant adenovirus vectors carrying genes of green fluorescent protein (Ad-GFP) and MG53(Ad-MG53) or recombinant adenovirus of silencing endogenous MG53 expression (Ad-MG53-RNAi) and oligonucleotides of adenovirus (Ad-ShRNA) as contrast. After 48 hours Transfection, the level of MG53 expression was quantified by Western blot analysis in order to make clear the upregulation or downregulation of the protein of MG53. Then, the function of ion channels、electrophysiological properties and involving mechanism were studied.Part 1:Effects of changes in the expression of MG53 on the function of ion channel and electrophysiological properties in cardiomyocytesHere, we changed the expression of MG53 using adenovirus vector and siRNA interference technology in cultured cardiac myocytes to investigate the role of MG53 protein in cardiac electrophysiology. The results showed that:1. The regulation of MG53 on the function of Ito1.1. Effects of MG53 on density and dynamics of Ito current on cardiomyocytesNeonatal rat myocardial cells were transfected with four different adenovirus, then we recorded the Ito current after 48h. The results showed that the current density in the Ad-MG53 group were significantly increased than the Ad-GFP group, an increase of 1.1 times (P=0.000<0.05). That is to say, overexpression of MG53 can significantly increased the current density of Ito. Compared with Ad-ShRNA group, the current density in Ad-MG53i-RNAi group were significantly decreased by 38%(P=0.000<0.05). That is to say, Lower expression of MG53 could significantly decreased the current density of Ito. In four groups, the activation kinetics of Ito channel had no significant changes. the difference about the time to peak (TTP) at 60mV was not statistically significant. Meanwhile, overexpression of MG53 can significantly slow down Ito channel deactivation kinetics, Ito channel current decay time become longer. T0.5 in Ad-MG53 group was significantly longer than the Ad-GFP group (P=0.04<0.05). Lower expression of MG53 could significantly speed up Ito channel deactivation kinetics, Ito channel current decay time became shorter. TO.5 in Ad-MG53-RNAi group was significantly shorter than the Ad-ShRNA group (P=0.03<0.05). That is to say, MG53 could influence inactivation kinetics of Ito in NRVM.Adult rat ventricular myocytes were transfected with four different adenovirus, then we recorded the Ito current after 48h. The results showed that the current density in the Ad-MG53 group were not significantly increased than that in the Ad-GFP group. However, compared with Ad-ShRNA group, the current density in Ad-MG53i-RNAi group was significantly decreased by 25.4%(P=0.001<0.05). That is to say, Lower expression of MG53 can significantly decreased the current density of Ito in ADVM. In four groups, the activation kinetics of Ito channel had no significant changes. the difference about the time to peak (TTP) at 60mV was not statistically significant. Meanwhile, overexpression of MG53 could significantly slow down Ito channel deactivation kinetics, Ito channel current decay time became longer. T0.5 in Ad-MG53 group was significantly longer than the Ad-GFP group (P=0.008<0.05). Lower expression of MG53 could significantly speed up Ito channel deactivation kinetics. Ito channel current decay time became shorter. TO.5 in Ad-MG53-RNAi group was significantly shorter than that in the Ad-ShRNA group (P=0.02<0.05). That is to say,MG53 could influence inactivation kinetics of Ito in ARVM.1.2. Impacts of MG53 on activation of voltage-dependent Ito channelNRVM:The Ito current density of Ad-MG53 group were significantly higher than that of Ad-GFP group in the +0～+70 mV voltage range (P<0.05).The voltage-dependent activation curve had significant changes between the two groups. V0.5, act were 34.26±1.85 mV and 27.61 ± 1.99 mV, P=0.03<0.05, k values also had no significant difference between the two groups, which indicate that MG53 did not change the ion selectivity of Ito. The Ito current density of Ad-MG53-RNAi group were significantly lower than that of Ad-GFP group in the+10-+70 mV voltage range (P<0.05).The voltage-dependent activation curve had significant changes between the two groups. V0.5,act were 32.68±2.19 mV and 42.04±3.15 mV, P=0.03<0.05, k values also had no significant difference between the two groups, which indicate that MG53 did not change the ion selectivity of Ito.ARVM:The Ito current density of Ad-MG53 group were not significantly increased than that of Ad-GFP group in the -40～+70 mV voltage range (P >0.05).The voltage-dependent activation curve had not significant changes between the two groups. V0.5,act were 29.97±1.84 mV and 34.05±2.09 mV, P=0.15>0.05, k values also had no significant difference between the two groups, which indicate that MG53 did not change the ion selectivity of Ito. The Ito current density of Ad-MG53-RNAi group were significantly lower than that of Ad-GFP group in the +20-+70 mV voltage range (P<0.05).The voltage-dependent activation curve had not significant changes between the two groups. V0.5,act were 33.80±1.23 mV and 33.11 ±1.61 mV, P=0.74>0.05, k values also had no significant difference between the two groups, which indicated that MG53 did not change the ion selectivity of Ito.1.3. Impact of MG53 on inactivation of voltage-dependent Ito channelNRVM:Compared with Ad-GFP group, the voltage-dependent inactivation curve of Ad-MG53 group occurs a positive shift, k value of the differences between the two groups was not significant. Meanwhile, the voltage-dependent inactivation curve of Ad-MG53-RNAi group occured no shift, k value of the differences between the two groups was not significant.ARVM:Compared with Ad-GFP group, the voltage-dependent inactivation curve of Ad-MG53 group occurs no shift, k value of the differences between the two groups was not significant. Meanwhile, the voltage-dependent inactivation curve of Ad-MG53-RNAi group occured a negative shift, k value of the differences between the two groups was not significant.2. APD20, APD50 and APD90 in Ad-MG53 group was significantly shorter than the Ad-GFP group. RMP did not change between the two groups. APD20 and APD50in Ad-MG53-RNAi group was significantly longer than that in Ad-ShRNA group, while the RMP had not changed.Part 2:the role of MG53 in the regulation of calcium signaling in ARVMIn single cardiomyocyte, the changes of APD (the action potential duration) affect the inflow Ca2+, which would influence excitation-contraction coupling. in turn, it would affect the Systolic and diastolic function of the heart. Here, we changed the expression of MG53 using adenovirus vector and siRNA interference technology in cultured cardiac myocytes to investigate the role of MG53 protein in calcium signaling by confocal. The results showed that:overexpression of MG53 increased the level of calcium in cardiomyocyte in resting state、the transient and the contration. On the contrary, lower expression of MG53 decreased the level of calcium in cardiomyocyte in resting state、the transient and the contration.Part3:The mechanisms underlying MG53 regulation of the expression of cardiac auxilliary subunit KChIP21. Using a new generation large-scale high-throughput Sequencing technology platform (Hiseq2000),50 SE Sequencing strategy and Illumina Sequencing Kits v3, four groups of RNA samples(the Ad-GFP, Ad-MG53, Ad-ShRNA, Ad-MG53-RNAi) had been Hiseq Sequenced to find out the opposite trend in different group. it was found that MG53 regulated the expression of cardiac auxilliary subunit KChIP22. Effects of changes in the expression of MG53 on expression of Ito β subunits KChIP2The western blot results indicated that, adenoviral overexpression of MG53 increased KChIP2 protein levels in cardiomyocytes, whereas MG53 knockdown by RNA interference decreased KChIP2 protein expression,no matter in NRVM orARVM.3. Lower expression of MG53 promoted NF-kB shift to the nucleus4. A mutant plasmid of IκB-a which inhibited the phosphorylation of NF-κB was constructed, it was found that, IκB-α-mutant inhibited the down-regulation of KChIP2 and the up-regulation of NF-κB by the decrease of MG53. It gave us a hint that MG53 regulation of the expression of KChIP2 was related to the pathway of NF-κB.meanwhile,the inhibitor of NF-vB,BAY-117082, had the same effects just like IκB-α-mutant.5. From analysis of the level of MG53 expression,we found that, as the growth of age, the MG53 protein expression was increaseing,which was the same as the KChIP2.6. The expression of MG53 in different organs MG53 was not expressed in liver and kidney, but it was abundant in heart and muscle, just as previously reported. However, MG53 was also expressed in the uterus and blood vessels.Conclusion:1. MG53 regulated the current density of transient outward potassium channels (Ito) and channel deactivation kinetics, but the channel activation kinetics, and voltage-dependent activation had no significant change.2. MG53 regulated the APD of ARVM.3. MG53 regulated the level of calcium in rest state,the transient and the contraction of ARVM.4. MG53 regulation of the expression of KChIP2 was related to the pathway of NF-κB.5. MG53 protein expression was increaseing with the growth of the age, which was the same as the KChIP2.6. MG53 was not only expressed in skeletal muscle and heart,but also in smooth muscle such as uterus and blood vessels.