| Backgrounds and Objectives:The function transport between cytoplasmic membrane (PM) surface and endplasmic/saroplasmic reticulum(ER/SR) is an important feature of all excitable cells while the JMCs (junctional membrane complexes) located between them is the structural basis to make its effective transport come true. Junctophilin (JP, JPH) is a kind of protein family associated with the formation of JMC and provides structural foundation for the signal communication between the cytoplasmic membrane and the endplasmic reticulum.JMC keeps intracellular Ca2+stability via coupling the ion channels between the membrane surface and the ER/SR. JPH is divided into four subtypes in mammals, namely JPH1, JPH2, JPH3and JPH4, but only JPH2is the one that mainly located in the cardiac muscle.Studies revealed that JPH2can facilitate the functional coupling between the voltaged-gate calcium channels(VGCCs) in the cytoplasmic membrane and ryanodine receptors(RyRs) in the ER/SR.Ryanodine receptors (RyRs) are the SR Ca2+release channel in eukaryotic cell. There are three subtypes in mammals, called RyR1, RyR2and RyR3. In the cardiac myocytes, the excitation-contration coupling relies on RyR2calcium release channel-induced calcium release (CICR).Small conductance Ca2+activated K+channels (SK channels) are the voltage-independent K+ion channels that activated by intracellular Ca2+, and exist in nearly all excitablel mammal cells. They can be divided into four subtypes according to the different pharmacological propeqQRTies of apamin, namely SK1、SK2、SK3and SK4. The SK2channle located in the membrane of cardiac myocytes takes part in making up the action potential repolarization and controls the rhythm of cardiac conducting tissue. Previous study in our laboratory showed the functional relationship between the SK2channle and RyR2operated calcium release channle.Little is known the mechanism of this coupling. We wonder whether the functional cross-talk between the RyR2and the SK2channle in the cardiac myocyte is related to JPH2. In order to document this hypothesis, we silenced JPH2in the neonatal cardiac myocytes (NCMCs) using RNAi and determined the effect of lentiviral-mediated siRNA JPH2on the SK2and RyR2in the NCMCs for fuqQRTher study on the molecular mechanism of the coupling of the SK2channle to the RyR2.1、Two sequences (21nt) targeted mouse JPH2mRNA were designed according to the software.The recombinant lentivirul vectors mediated JPH2-shRNA were constructed. The recombinants were identified by PCR, and the positive clones identified by sequencing. The recombinant lentiviral vectors were packaged and amplified in293T cells. The recombinant lentivirus products were defined as Lenti-siJPH2-1and Lenti-siJPH2-2.2、Culture of the NCMCs. The NMCMs were infected with the lentiviral vectors for48h. Infected cells were monitored by GFP fluorescence under the inverted microscope. The infection efficiency of the neonatal mouse cardiac myocytes infected with the lentiviral-mediated siRNA vectors was determined by Flow cytometry.3、The expression of JPH2mRNA in the NMCMs infected with the lentiviral-mediated siRNA vectors was determined by qRT PCR. 4, The expressions of the SK2protein and RyR2mRNA in the NMCMs infected with the lentiviral-mediated siRNA vectors were documented by qRT-PCR and Western blotting.5、Statistical treatment:data were analyzed using the software SPSS17.0, choose a=0.05as test standard. Differences between groups were evaluated by one-way ANOVA followed by paired or unpaired Student’s T tests as appropriate.Results:1. The Lentiviral-mediated JPH2-shRNA vectors were successfully constructed2. The growed as single lyer in the culture medium and showed the cardiac beat after the culture of2-3days.3. The infection efficiency of the neonatal mouse cardiac myocytes treated with Lenti-NC、Lenti-siJPH2-1and Lenti-siJPH2-2was66.2%、66.5%,68.4%, separately, when MOI is100.4. The relative level of JPH2mRNA in the NMCMs infected with Lenti-NC. Lenti-siJPH2-1and Lenti-siJPH2-2is0.96±0.07,0.26±0.09and0.29±0.12. The expression of JPH2in the NMCMs infected with lentiviral-mediated siRNA vectors decreased compared with control siRNA vector, p<0.01.5. The relative level of SK2mRNA in the NMCMs infected with Lenti-NC、 Lenti-siJPH2-1and Lenti-siJPH2-2is1.26±0.05、0.08±0.02and0.29±0.12. The expressions of the SK2mRNA in the NMCMs infected with lentiviral-mediated siRNA vectors decreased cpmpared with control siRNA vector, p<0.01.6. Expression of the SK2protein in the NMCMs transfected with Lentiviral-mediated JPH2-siRNA vectors for60h were suppressed by Western blotting analysis.Conclusions: Two recombinant lentivirus vectors carried mouse JPH2-siRNA Lentiviral vectors were successfully constructed.Both Lenti-siJPH2-1and Lenti-siJPH2-2downregulated the expression of JPH2mRNA in the neonatal mouse cardiac myocyte. The knockdown of JPH2suppresses the expression of SK2channle. More research is ongoing. |
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