| Part I Subcellular localization of HCN4pacemaker channel in the sinus nodeObjective:toinvestigate the sub-cellular localization of hyperpolarization-activated cyclic nucleotide-gated channel4(HCN4) expressed in rabbit sinoatrial (SAN) cells and the effects of such localization on the characteristics of HCN4channel in SAN cells.Methods:The preparations of sinoatrial node cells were isolated from rabbit heartusing enzymatic method, membrane cholesterol depletion was achieved by methyl-β-cyclodextrin (MβCD) treatment. Then we used a discontinuous sucrose gradient and Western blot analysis to detect HCN4proteins in SAN lipid raft-enriched fractions and nonlipid raft membranes, a whole-cell patch clamp technique was employed to record If in isolated rabbit SAN pacemaker cells.Results:HCN4proteins localized to low-density membrane together with caveolin-3, which was the scaffolding protein of caveolae. Lipid raft disruption with MβCD changedthe specific HCN4localization. It also shifted the midpoint of activation of If in SAN cells to the positive direction. In accordance with an increased If, MβCD-treated SAN cells showed large increases of diastolic depolarization slope and rate. We also found that the kinetics of native f-channel deactivation were slower after lipid raft disorganization.Conclusions:These results indicate thatHCN4channel localize to lipid rafts and that disruption of lipid rafts causes channels to redistribute within the membrane and modifies their kinetic properties.Part II The changes of sinus node function and electrophysiological characteristics and caveolin-3, HCN4expression in rabbit heart failureObjective:The objective of this study was to investigate the change of function and electrophysiological properties in sinus node, and the change of mRNA levels and protein expressions of caveolin-3and HCN4in control group and heart failure group.Methods:HF was produced by combined aortic insufficiency and abdominal aortic constriction. Before modeling and after8weeks with echocardiography to detect whether the model was produced successfully. Then we use to test the changes of sinus node conduction time (SACT) and sinus node recovery time (SNRT). We detected the change of action potential with standard glass microelectrode technique. The change of caveolae in sinoatrial node tissue was observed by transmission electron microscopy. RT-PCR, western plot and immunohistochemistry were used to observe the mRNA levels and protein expression of caveolin-3and HCN4.Results:Compared with the control group, LVIDs, LVIDd, ESV, EDV values were lower in heart failure group (p<0.05), while the EF and FS values increased (p<0.05). HF group SACT, SNRT, cSNRT were significantly prolonged (p<0.05). MDP, APA, DDR, RPF values were reduced, while APD50, APD90were prolonged (p<0.05). mRNA and protein expression levels of caveolin-3and HCN4significantly reduced after heart failure, and HCN4abundance positively correlated with the reduction of caveolin-3.Conclusions:Reduction of caveolae number lead to the expression change of HCN4channel which location in caveolae, downregulation of HCN4expression might contribute to CHF induced sinus sinus node dysfunction,the reduction of HCN4may be associated with the change of caveolin-3proteins. These findings provide novel information about the molecular basis of normal and disease-related impairments of cardiac impulse formationPart Ⅲ The effects of caveolin-3on the hyperpolarization-activated cyclic nucleotide-gated channel heterologously expressed in Xenopus oocytesObjective:To study the effects of caveolin-3on human HCN4channel heterolog-ously expressed in Xenopus oocytes.Methods:We transcribed human HCN4, Cav-3and p104L DNA into complementary using mMESSAGE mMACHINE(?) T7Kit, then the cRNAs were injected in enzymatically isolated Xenopus oocytes.The experiment was divided into five groups:HCN4, HCN4+Cav-3, Cav-3, HCN4+p104L and p104L (as negative control) group. Human HCN4currents were recorded using two-electrode voltage clamp technique.Results:The human HCN4currents were successfully expressed and recorded, with characteristics as follows:1) the channel open in a time-dependent manner after a series of hyperpolarization voltages;2) the more hyperpolarizated, the faster the channel activated;3) zatebradine and CsCl significantly inhibited the channel.4) Cav-3increased the hHCN4current amplitude, accelerated the activation and deactivation kinetics without affecting the voltage dependence and reversal potential. P104L caused a two-fold increase in the activation time constant for IHCN4and shifted the voltage of the steady-state inactivation to a more negative potential.Conclusions:Cav-3and HCN4gene, co-expressed in Xenopus oocytes, can regulate the electrophysilological properties of HCN4channel, and may have a physiological correlation. |
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