Rbfox2 Regulates The Alternative Splicing Of Ca_V1.2 Calcium Channel:Implications In Hypertension

Background:Voltage-gated L-type calcium channel Ca_V1.2 triggers the calcium influx in vascular smooth muscle cells,which is important for maintaining myogenic tension and blood pressure.Alternative splicing,as a post-transcriptional mechanism,optimize the functions of Ca_V1.2 calcium channel.RNA-binding protein Rbfox2 is reported to regulate the alternative splicing of Ca_V1.2 calcium channel in the neural development.However,the roels of Rbfox2 in the regulation of Ca_V1.2 calcium channel and pathogenesis of hypertension is still undefined.Objective:Our study aims to investigate the expression of Rbfox2 and Ca_V1.2calcium channel alternatively spliced exons exon 9*and 33 in hypertensive arteries,and to detect Rbfox2’s effects on the function of Ca_V1.2 calcium channel and vascular myogenic tone.Methods:First,the expression of Ca_V1.2 calcium channel alternative exons 9*and 33,and Rbfox2 are investigated by clony RT-PCR and Western blotting in rat mesenteric arteries,respectively.Second,we study the Rbfox2’s effects on regulation of Ca_V1.2 alternative splicing and electrophysiological properties by RT-PCR and whole-cell patch-clamp respectively.Finally,vascular myogenic tone is also determined by Living Systerm after knocking down Rbfox2 expression of rat mesenteric artery.Results:We find that Ca_V1.2 channel with exon 9*is increased by 10.3%,whereas Ca_V1.2 channel with exon 33 is decreased by 10.5%.However,the expression level of Rbfox2 is increased about 3 folds,which could be attributed to the dominant-negative isoform of Rbfox2.In vascular smooth muscle cells,we uncover that knockdown of Rbfox2 can increase exon 9*inclusion,whereas decrase exon 33inclusion.In vascular smooth muscle cells,siRNA-mediated knockdown of Rbfox2shifs Ca_V1.2 channel’steady-state activation(SSA)and steady-state inactivation(SSI)curves to hyperpolarization,which leads to the window current potential become more negative.Moreover,the diminished-Rbfox2 enhances the pressure-induced vascular myogenic tone of rat mensenteric artery.Conclusions:Our study indicates that Rbfox2 modulates the function of vascular Ca_V1.2 calcium channel by dynamically regulating the alternative splicing of Ca _V1.2calcium channel,which influence the vascular myogenic tone.Importantly,we elucidate the underlying mechanisms of enhanced Ca_V1.2 channel activity in hypertensive arteries.Therefore,this work might provide a potential therapeutic target in the management of hypertension.