The Mechanism Of Muscarinic Receptor And Big-conductance Voltage-and Ca²⁺-activated K⁺ Channel On The Phenotypic Change Of Bladder Smooth Muscle Cells Under Mechanical Stress

Objective:The bladder realizes its function through the dilation during the urine storage period and the contraction during the urination period.In this process,the functional phenotype of bladder cells will change accordingly in response to different biological stress environments.The previous research of our group found that there are a series of receptors and signal pathways that mediate effects of mechanical stress in bladder smooth muscle cells and urothelial cells,which participate in the process of biomechanical signal transduction,cell proliferation,contraction,extracellular matrix metabolism,and cytoskeleton changes.Among them,the contraction phenotype is the basis for the normal function of bladder.Bladder outlet obstruction（BOO）is a common disease in urology,which usually causes urine retention in the bladder,excessive stretching of the bladder wall,and inflammatory reactions.In severe cases,it can cause fibrosis and irreversible pathological changes in the structure and function of bladder.In the early stage of BOO,the compensatory hypertrophy and compensatory increase in contractility of bladder smooth muscle are occurred to overcome the increased resistance of urination.If bladder emptying continues to be blocked,the contractility of bladder detrusor decreases,and the bladder function is decompensated.Therefore,bladder smooth muscle cells and their phenotypic changes play an important role in BOO-induced pathological bladder remodeling.The use of anticholinergic drugs to treat the symptoms of overactive bladder in patients with benign prostatic hyperplasia has a certain effect and does not increase the incidence of urinary retention.The main function of muscarinic receptors is to regulate the contraction and relaxation of smooth muscle cells.But in our previous studies,we found that the function of muscarinic receptors is also directly involved in the regulation of cell proliferation,extracellular matrix metabolism and inflammation under mechanical stress.In addition,treatment with anticholinergic drugs has side effects.Patients may experience dry mouth,dry eyes,dry skin,vomiting,tachycardia,constipation,abdominal pain,drowsiness,headache,and paresthesia.Therefore,it is of great significance to find a new target that mediate the phenotypic changes of bladder smooth muscle cells.Big-conductance voltage-and Ca²⁺-activated K⁺（BK）Channel regulates cell excitability and contractility.At present,the research on the function of BK channel in bladder smooth muscle cells mainly focuses on contractile function,and less research pays attention to its effect on the phenotypic transformation of bladder smooth muscle cells.In vascular smooth muscle,BK channel may play an important role in the pathological remodeling of blood vessels caused by hypertension.Whether the BK channel also regulates the process of bladder biological stress-induced signal transduction,requires further experimental research.Previous studies have found that activation of muscarinic receptor in bladder smooth muscle cells can inhibit BK channel activity by depleting Ca²⁺in the sarcoplasmic reticulum.In vascular smooth muscle cells,Ca²⁺is not only involved in cell contraction,but also be related to genetic transcription.The realization of this function is related to the Ca²⁺/calmodulin-induced phosphorylation of c AMP response element-binding protein（CREB）.When the intracellular Ca²⁺concentration is reduced,the phosphorylation of CREB is inhibited,and thereby adjusting the expression of vascular smooth muscle cell differentiation markers.In addition,the muscarinic receptor and BK channel are closely related to the changes of intracellular Ca²⁺concentration.In summary,in order to explore the role of muscarinic receptors and BK channel in the phenotypic changes of bladder smooth muscle cells under biological stress,this study first established a partial bladder outlet obstruction（PBOO）rat model to observe PBOO effect on the phenotype of bladder smooth muscle.Further in vitro cell experiments were conducted to study the effects of biological stress,muscarinic receptors and BK channel on the expression of bladder smooth muscle cells contraction-related proteins and related mechanisms.Methods and materials:Our study could be divided into the following five chapters.Chapter I:To study the effect of excessive biological stress on the phenotypic changes of bladder smooth muscle,we established a PBOO rat model by partially ligating the bladder neck of Sprague Dawley rats.And we performed m RNA whole-genome gene microarray sequencing analysis on rat bladder smooth muscle.After screening the differential genes,we performed GO enrichment analysis and gene set enrichment analysis（GSEA）.According to the enrichment analysis results,the contraction-related protein genes and BK channel genes were further screened for differential analysis.At the same time,Masson staining and immunohistochemical staining were used to observe the changes of rat bladder structure under PBOO and the expression changes of BK channel on bladder smooth muscle cell membrane under PBOO.Chapter II:In order to explore the effect of biological stress on the contraction phenotype of human bladder smooth muscle cells（HBSMCs）,we applied cyclic hydrostatic pressure to them and exposed them to 200cm H₂O hydrostatic pressure for 2 h,6 h and 24 h.The control group was not subjected to hydrostatic pressure treatment.RT-PCR and WB were used to detect the expression changes of cellular contraction-related proteins（α-Actin,SM22α,Calponin and Myosin heavy chain）and BK channel subunits（BKα,BKβ1 and BKβ4）in HBSMCs.The cell contraction array kit was used to detect the changes of contraction function in HBSMCs under hydrostatic pressure.And the contraction index was calculated with the collagen area change.Chapter III:In order to explore the effect of muscarinic receptor activation on the expression of contraction-related proteins and BK channel subunits in HBSMCs,we used 1μM,10μM and 100μM concentration of carbachol to stimulate HBSMCs.RT-PCR and WB were used to detect the expression of contraction-related proteins and BK channel subunits.Furthermore,we used selective M₂ receptor inhibitor AF-DX116 or M₃ receptor inhibitor 4-DAMP to study the role of muscarinic receptor in hydrostatic pressure-induced HBSMCs contraction phenotype changes and BK channel subunit expression changes.The collagen gel method was used to detect the changes of contractility of HBSMCs regulated by muscarinic receptor under hydrostatic pressure.Finally,we used PKC kinase activity detection assay,PKC agonist and ryanodine receptor（Ry R）inhibitor to explore the mechanism of muscarinic receptor-mediated BK channel expression in HBSMCs under hydrostatic pressure.Chapter IV:In order to study the mechanism of muscarinic receptor and BK channel regulating the contractile phenotype of bladder smooth muscle cells,we used the BK channel antagonist Paxilline and the agonist NS1619 to stimulate HBSMCs.RT-PCR,WB and cell contraction assay were used to detect whether the BK channel mediates the expression of contraction-related proteins and the contractility in HBSMCs under hydrostatic pressure.Finally,the voltage-gated Ca2+channel antagonist Nifedipine,Ry R antagonist Ryanodine,and inositol triphosphate receptor antagonist Xestospongin-C were used to block the main sources of intracellular Ca2+.And then the expression changes of contraction-related proteins in HBSMCs were detected under the stimulation of muscarinic receptor agonist or BK channel antagonist.Chapter V:To explore the expression changes of muscarinic receptor,BK channel and contraction-related proteins during the growth of bladder smooth muscle cells under physiological stress stimulation,we used the small intestine submucosa（SIS）acellular scaffold to construct mouse bladder defect repair models.And the changes of the SIS scaffold at 1 week,3 weeks and 6 weeks after the operation were observed by Masson staining of the mouse bladder.In addition,we used single-cell sequencing to analyze the changes of cell population in SIS scaffold after the operation.Differential expression analysis of muscarinic receptor,BK channel and contraction-related protein genes in bladder smooth muscle cells was also performed.Results:Chapter I:On the basis of successful constructed PBOO rat model,GO enrichment analysis showed that the biological processes related to muscle tissue development,muscle contraction,and muscle regulation in the bladder smooth muscle of PBOO rats were significantly down-regulated.The cellular components related to the actin skeleton and contractile fibers were significantly down-regulated.In terms of molecular functions,gene expressions related to Ca²⁺binding and voltage-gated cation channel activity were significantly down-regulated,but gene expression related to G protein-coupled receptor binding was significantly up-regulated.In the results of GSEA,differential genes were also enriched in pathways related to actin cytoskeleton and Ca²⁺signaling.The expression of bladder smooth muscle contraction-related protein genes in PBOO rats was down-regulated（Myh11-2.90,P=0.004;Cnn1-1.67,P=0.0005;Tagln-1.37,P=0.0004）.Similarly,BK channel subunits gene expression in PBOO rat bladder smooth muscle was significantly down-regulated（Kcnma1-3.22,P=0.04;Kcnmb1-1.92,P=0.0003）.Immunohistochemical staining showed that the number of BK channels on the bladder smooth muscle cell membrane of PBOO rats was significantly reduced compared with the sham operation group.Chapter II:After HBSMCs were exposed to hydrostatic pressure of200cm H₂O for 2 h,6 h,and 24 h,RT-PCR detection showed that the expression of contraction-related protein genes was highest in the 2 h group（α-Actin:1.47±0.31,P<0.05;Calponin:1.70±0.31,P<0.05;Myosin HC:3.15±0.49,P<0.01）,except for that the expression of SM22αgene was highest in the 6 h group（3.92±0.61,P<0.01）.In addition to theα-Actin gene,the expression of other contraction-related protein genes gradually decreased with the extension of treatment time,and was significantly lower than the control in the 24 h group（SM22α:0.69±0.03,P<0.05;Calponin:0.38±0.05,P<0.01;Myosin HC:0.33±0.03,P<0.01）.The WB results were consistent with the RT-PCR results.The gene expression of BK channel subunits in HBSMCs under hydrostatic pressure showed a similar trend.The gene expression of all subunits reached the highest in the 2 h group,and BKβ4 was the most up-regulated subunit（3.32±0.76,P<0.01）.With the extension of treatment time,the gene expression of BK channel subunits gradually decreased,reaching a minimum at 24 h.Except for the BKαsubunit,the expressions of BKβ1（0.31±0.11,P<0.01）and BKβ4（0.31±0.06,P<0.01）in the 24 h group were significantly lower than those in control group.The cell contraction assay found that the contractility of HBSMCs was significantly enhanced in the 2 h group and 6 h group（contraction index:control:31.43±2.10%;2 h:66.05±4.89%;6 h:60.10±3.83%）.Chapter III:The expression levels of contraction-related protein genes all increased most significantly in the 1μM carbachol group（α-Actin:1.53±0.19,P<0.05;SM22α:1.78±0.30,P<0.05;Calponin:1.89±0.26,P<0.05;Myosin HC:2.29±0.45,P<0.05）.Similarly,the expression levels of BK channel subunit genes reached the highest level in the 1μM group（BKα:1.67±0.19,P<0.05;BKβ1:4.28±0.59,P<0.01;BKβ4:3.66±0.69,P<0.01）.The WB results verified that 1μM was the optimal concentration of carbachol.Compared with the hydrostatic pressure group,activation of muscarinic receptor can further promote the expression of Calponin（1.98±0.27,P<0.05）and Myosin HC（2.83±0.36,P<0.05）,while antagonists of M₂ and M₃ receptors can reduce the expression of contraction-related protein Calponin（4-DAMP:0.51±0.08,P<0.05;AF-DX116:0.49±0.07,P<0.05）and Myosin HC（4-DAMP:0.32±0.05,P<0.01;AF-DX116:0.45±0.05,P<0.05）under hydrostatic pressure.Carbachol can increase the contraction index of HBSMCs under hydrostatic pressure,but there was no significant difference between carbachol group and hydrostatic pressure group（74.03±7.56%vs 66.05±4.89%,P>0.05）.M₃ receptor antagonist can significantly inhibit the contractility of HBSMCs under hydrostatic pressure（46.33±8.08%vs 66.05±4.89%,P<0.05）,while M₂ receptor antagonist also show a certain inhibitory effect（55.33±5.03%vs 66.05±4.89%,P>0.05）.The activation of muscarinic receptor can promote the gene expression of BK channel subunits in HBSMCs under hydrostatic pressure（BKα:1.63±0.12,P<0.05;BKβ1:3.56±0.21,P<0.01;BKβ4:3.41±0.22,P<0.01）.Both M₂ and M₃receptor antagonists can significantly reduce the expression of BK channel subunit genes under hydrostatic pressure.At the protein level,changes in protein synthesis showed the same trend.The results of PKC kinase activity assay showed that carbachol and PKC agonist PMA can significantly promote PKC kinase activity.And M₃ receptor antagonist can significantly inhibit hydrostatic pressure-induced PKC kinase activity.PMA can significantly up-regulate the expression of BKαand BKβ4 genes（BKα:2.07±0.50,P<0.05;BKβ4:1.55±0.33,P<0.01）.The PMA structural analogue 4αPMA has no such effect.Similarly,the WB results showed the same changing trend.After using Ry R antagonist,the effect of promoting the up-regulation of BK channel subunit gene expression in PMA disappeared.Chapter IV:The BK channel antagonist Paxilline can significantly promote the expression of contraction-related protein genes in HBSMCs（m RNA:α-Actin:1.40±0.14,P<0.05;Calponin:1.59±0.12,P<0.05;Myosin HC:1.58±0.22,P<0.05）,while the BK channel agonist NS1619 had no significant effect on the contraction phenotype of HBSMCs.The changes of BK channel activity had no significant effect on the contractile function of HBSMCs（Paxilline:66.97±4.99%vs 66.05±4.89%,P>0.05;NS1619:53.67±4.51%vs 66.05±4.89%,P>0.05）.After blocking intracellular Ca²⁺sources,both RT-PCR and WB detection showed that the muscarinic receptor agonist and the BK channel antagonist Paxilline had no effect on the expression of HBSMCs contraction-related protein under hydrostatic pressure,which preliminarily indicated that intracellular Ca²⁺signal was involved in the regulation effects of muscarinic receptors and BK channel on the contraction phenotype of HBSMCs.Chapter V:Masson staining showed that as the postoperative time increased,more and more cells grew in the SIS scaffold.At 3-week after the operation,epithelial cells covered the SIS patch,and neovascularization occurred at 6-week after the operation.Our cluster analysis found that with the extension of postoperative time,the cluster distribution of cells in the SIS scaffold and the control group gradually overlapped,indicating that the cell types in the SIS scaffold gradually approached the control group.After the classification of cells were identified,we found that smooth muscle cells began to growth in the 3-week group and gradually increased.Furthermore,the differential expression analysis of muscarinic receptors,BK channel,and contraction-related protein genes between the bladder smooth muscle cells in the SIS scaffold and the bladder smooth muscle cells in the control group,we found that the muscarinic receptors,BK channel subunits and contraction-related protein genes were significantly down-regulated in the 3-week group.Six-week after the operation,the gene expression of muscarinic receptors and BK channel subunits was up-regulated compared with the sham group,and the gene expression of contraction-related protein was similar to that of the sham group.Conclusions:After the analysis of the results,the subject mainly draws the following conclusions:1.In this part,the PBOO rat model was successfully constructed and its genome-wide gene microarray was sequenced.We found that the contraction phenotype of bladder smooth muscle in PBOO rat was significantly changed,and the expression of contraction-related protein genes was significantly down-regulated.As one of the voltage-gated cation channels,BK channel has similar changes during this process,suggesting that BK channel may be involved in the contractile phenotypic change of rat bladder smooth muscle.2.Cyclic hydrostatic pressure can cause changes in the contractile phenotype of HBSMCs.Short-term hydrostatic pressure exposure can enhance cell contractility.With the prolonged exposure time,cell contraction ability gradually decreases and the expression of contraction-related protein genes decreases.The expression of BK channel subunits in HBSMCs under hydrostatic pressure showed similar changes,suggesting that it may participate in the structural and functional changes of HBSMCs under cyclic hydrostatic pressure.3.Both M₂ and M₃ receptors are involved in the regulation of phenotypic changes and contractile function of HBSMCs caused by hydrostatic pressure.In addition,the M₃ receptor mediates the change of BK channel subunit expression in HBSMCs under hydrostatic pressure by promoting the inhibitory effect of PKC kinase on the Ry R.4.Inhibition of BK channel activity can promote the expression of contraction-related proteins in HBSMCs,and BK channel antagonist may improve the contractile phenotype of BOO bladder smooth muscle.As an important second messenger in cells,Ca²⁺mediates the process that muscarinic receptor and BK channel regulate the contraction phenotype of HBSMCs under hydrostatic pressure.5.In the mouse bladder repair model,the expression of muscarinic receptor,BK channel and contraction-related protein genes have a relatively synergistic change during the growth of neonatal bladder smooth muscle cells.Muscarinic receptor and BK channel may be involved in the growth process of bladder smooth muscle cells under physiological stress stimulation.