Extracellular Calcium Sensing Receptor Variants Increase Pulmonary Hypertension Susceptibility

Objective:Pulmonary hypertension（PH）is a serious disease,in which the extracellular calcium sensing receptor（Ca SR）is mechanistically important.On the basis of clinical studies,it’s found that the common variation of Ca SR（rs6776158（-167 G>A）,rs1048213（-213 T>C）,rs9883099（-175 C>A）,rs1042636（c.2968 A>G））were related to the severity and susceptibility of patients with idiopathic pulmonary arterial hypertension（IPAH）.This study intends to further explore the effects of the above common variants of Ca SR on its transcriptional regulation and protein function at the cellular and animal levels,and then clarify its mechanism in the occurrence of IPAH.Methods:Dual luciferase Reporter Gene Assays were used to explore the effects of common promoters mutations rs6776158（-167 G>A）,rs1048213（-213 T>C）,rs9883099（-175 C>A）on Ca SR transcriptional activity.The PSIPRED 4.0 and Robetta tools were used to predict the effects of rs1042636（c.2968 A>G）,a common variant on the exon of Ca SR gene,on the secondary structure and three-dimensional conformation of Ca SR protein,respectively.The HEK293 cells were treated with different concentrations of extracellular calcium solution,and then Fura-2/AM fluorescent probe and fluorescence imaging system were used to dynamically detect intracellular calcium concentration([Ca²⁺]_i)to determine the activity of Ca SR of different genotypes.Ca SR 991G point mutant rats were constructed by CRISPR/Cas9 technique.Male SD rats aged 8 weeks were exposed to hypoxic chamber（10%O₂）after a single injection of 20 mg/kg Sugen5416 for2 weeks to replicate Su Hx-PH model.The mean pulmonary pressure（m PAP）was measured by Power Lab/4SP data acquisition system.The cardiac output（CO）were measured by thermodilution method.Pulmonary vascular resistance（PVR）was calculated using the formula m PAP/CO and right ventricular hypertrophy index was calculated by ratio of the weight of right ventricle to the sum of the weight of the left ventricle and septum（RV/（LV+S））.The vasoconstriction levels of different Ca SR genotypes of pulmonary arteries were detected after hypoxia by pulmonary arterial tension test.Western blotting was used to detect the expression of Ca SR protein in HEK293 cells and also in lung tissues of PH rat models with different genotypes.The localization and expression of Ca SR both in HEK293 cells and in pulmonary artery smooth muscle tissues of PH rats with different genotypes were detected by immunofluorescence.Cell Counting Kit-8（CCK8）and Ed U detection Kit were used to detect the proliferation ability of PASMCs of PH rats with different Ca SR genotypes.The migration ability of PASMCs of different Ca SR genotypes was detected by scratch healing experiment.Results:（1）The common mutation rs6776158（-167 G>A）,rs1048213（-213 T>C）and rs9883099（-175 C>A）on the Ca SR gene promoter increased the transcription activity of Ca SR in reporter gene assay.（2）The minor allele 2968G of the common exon variant rs1042636 induces a substitution of arginine（R）with glycine（G）at position 990 of Ca SR.The secondary structure of Ca SR was predicted online by PSIPRED 4.0,showing the spiral break between codon 987 and 988,which change the c-terminal and tail structure of Ca SR protein in cells.（3）Using Robetta tool to predict the three-dimensional structure of Ca SR in different genotypes,the common variant rs1042636（c.2968 A>G）on the exon broke the hydrogen bond between 987M and 990R residues,significantly affecting the three-dimensional configuration of the Ca SR protein domain（972-997aa）.（4）The expression of Ca SR protein of different genotypes in HEK293 cells was detected by Western blotting,and the results showed that the expression of mutant 990G and wild-type 990R protein of Ca SR were increased in HEK293 cells,and the expression were mainly increased in the form of polymers.Immunofluorescence staining showed that Ca SR mutant 990G protein and wild-type 990R protein were normally expressed on cell membrane.The sensitivity of Ca SR was evaluated according to the concentration for 50%of maximal effect(EC₅₀)in response to extracellular calcium stimulation in cells of different genotypes Ca SR.The results showed that under continuous stimulation with different concentrations of extracellular calcium solution, the sensitivity of HEK293 cells transfected with mutant 990G to extracellular calcium stimulation was enhanced compared with that of wild-type 990R(EC₅₀3.00±0.04 m M vs 5.41±0.09 m M)(P=1.74×10^-32).（5）The point mutant SD rats（Ca SR p.R991G）corresponding to rs1042636（c.2968 A>G） mutation found in IPAH population were successfully constructed,including heterozygous（A/G）and homozygous（G/G）mutant rats.In the replication Sugen/ hypoxia-induced pulmonary hypertension rat model,m PAP and PVR were significantly increased in the point mutation rats compared with wild-type rats, suggesting that PH phenotype was significantly aggravated,and there was no significant difference between heterozygous and homozygous mutant SD rats.（6）Western blotting results showed that in Su Hx-PH SD rats,compared with wild-type rats,Ca SR p.R991G point mutation significantly increased the protein expression of Ca SR in lung tissues.Immunofluorescence staining also showed that Ca SR point mutation significantly increased the expression of Ca SR in pulmonary artery smooth muscle of PH model rats.There was no difference in Ca SR expression between heterozygous and homozygous mutant rats.（7）Intracellular calcium concentration([Ca²⁺]_i)was significantly increased in r PASMCs of mutant SD rats treated with 6 m M extracellular calcium concentration([Ca²⁺]_o) compared with wild-type cells both in hypoxic（1%O₂）and in normoxic（21%O₂） conditions（P<0.05）.The hypoxia-induced vasoconstriction response of pulmonary arteries（PAs）isolated from mutant rats（Ca SR p.R991G）was stronger than that of wild-type rats.There was no significant difference in Ca SR activity between heterozygous and homozygous mutant rats.（8）CCK8,Ed U assay and wound healing test showed that Ca SR p.R991G point mutation promoted the proliferation and migration of r PASMCs（P<0.05）.There was no significant difference between heterozygous and homozygous mutant rats.Conclusions:The common mutation rs6776158（-167 G>A）,rs1048213（-213 T>C）and rs9883099（-175 C>A）on the Ca SR gene promoter increased the transcription activity of Ca SR gene.The common variant rs1042636（c.2968 A>G）on the exon increased the protein expression and sensitivity of Ca SR that may ultimately enhance the susceptibility to IPAH developmen through elevation Ca SR-mediated[Ca²⁺]_iin PASMCs,and aggravated the the severity of PH in SD rats.These findings may benefit clinical prognosis and treatment for IPAH.