Neuropeptide W And Rbfox1/2 Protein Regulate Cav1.2 Calcium Channel Of Vascular Smooth Muscle

Hypertension is a chronic disease with stably elevated arterial blood pressure,which could induce many cardiovascular diseases like atherosclerosis,stroke,myocardial hypertrophy and heart failure.L-type Cav1.2 calcium channel plays an important role in the arterial constriction,its dysfunction is closely related to the hypertension.The functions of Cav1.2 calcium channel can be regulated by many mechanisms,including G protein-coulped receptors(GPCRs)and alternative splicing(AS).These regulatory mechanisms can make subtle modulation of vascular Cav1.2 function,then regulate vascular smooth muscle constriction and participate in the development of hypertension.Part 1:Modulation of Cav1.2 calcium channel by neuropeptide W regulates vascular myogenic tone Background:NPW,an endogenous ligand for GPR7,was first found to play important roles in central nerve system,it was also involved in the regulation of intracellular calcium homeostasis via L-type calcium channels,but whether it affects the functions of vascular Cav1.2 channel and blood vessels is still unknown.Objective:This study aimed to clarify the regulatory mechanisms how NPW and regulate the functions of vascular Cav1.2 calcium channel,and their possible roles in vascular hypertension were also investigated.Methods:First,in order to investigate the pathological relevance,we detected the expression level of NPW mRNA in normotensive and hypertensive arteries.Second,we used NPW-23,an active form of NPW,to study its effect on Cav1.2 calcium channel function.Living system was used to validate its physiological functoion in maintaining small arterial myogenic tone.Results:Our study showed that the expression of NPW mRNA in hypertensive arteries was decreased when compared with normotensive ones.GPR7,the receptor of NPW,was coexpressed with Cav1.2 channels in arterial smooth muscle,and NPW-23 increased Ica,L of Cav 1.2 via binding to GPR7.Furthermore,NPW-23 enhanced vascular myogenic tone of mesenteric arteries.All of these suggested that NPW-GPR7 played an important role in hypertension.Part 2:Rbfox1/2 protein dynamically regulates the alternative splicing of Cay1.2 calcium channelBackground:As an important mechanism of post-transcriptional modulation for Cavl.2 calcium channels,alternative splicing(AS)has been found to involve in the neural development;and this process is regulated by the splicing factor Rbfox1/2.However,whether Rbfoxl/2 proteins participate in the splicing regulation of Cav1.2 calcium channel of hypertensive arteries remains unclear.Objective:This study aimed to clarify the regulatory mechanisms how Rbfoxl/2 proteins regulate the functions of vascular Cavl.2 calcium channel,and their possible roles in vascular hypertension were also investigated.Methods:We detected the expression of splicing variants of Cavl.2 channel,exon 9*and exon 33,and splicing factor Rbfoxin normotensive and hypertensive arteries by transcriptional scanning technique and western blotting,respectively.Molecular biological techniques like RNAi was used to study the effects of Rbfoxl/2 proteins on the Cavl.2 spliced exons,exon 9*and exon 33.Whole-cell patch clamp was applied to study the effects of these exons on electrophysiological function of Cavl.2 calcium channel.Results:When compared with normotensive arteries,the expression of exon 9*was increased in hypertensive arteries,while exon 33 was decreased.The splicing factor Rbfoxl/2 was expressed at vascular smooth muscle;more importantly,the expression of Rbfoxl/2 protein was also altered in the hypertensive arteries,implying its roles in the pathological process of hypertenison.Our in-vitro studies indicated Rbfoxl/2 proteins dynamically inhibited the alternative splicing of exon 9*,while enhanced the exon 33.Patch clamp studies demonstrated that the I-V curve and window currents potential of Cav1.29*,Δ33 showed the hyperpolarized shift when comparing with Cav1.2Δ9*,33,meaning that the opening and inactivation potential of Cavl.2 calcium channel were closer to the resting membrane potential,which will increase the activities of Cav1.2 calcium channel under phyciological condition.These data suggested that the dynamical regulation of alternative splicing of Cavl.2 calcium channel by Rbfoxl/2 proteins might directly make some roles in the development of hypertension.ConclusionsIn summary,NPW and Rbfoxl/2 proteins regulate Cavl.2 calcium channel function through differetnt mechanisms,which then control the vacular constriction;these may have a far-reaching impact on the development of hypertension.Our study will provide the new ideas to understand the pathogenesis of hypertension,and the novel therapeutic targets in the management of hypertension.