Role Of TRPM7/Smads Signaling Pathway In Remodeling Of Sinoatrial Node And Atrium In Rats With Sick Sinus Syndrome

Objectives: 1.To investigate the anatomic location and histomorphology characteristics of sinoatrial node in SD rat and establish stable sick sinus syndrome(SSS)model of SD rats.The morphological structure changes of sinoatrial node(SAN),the levels of angiotensin ?(Ang ?),transient receptor potential Melastatin 7(TRPM7),Smad2 and collagen content both in the SAN region tissue of SSS rats model were explored;2.To investigate the effect of AT1 receptor blocker losartan on TRPM7/Smad2 signaling pathway and synthesis of myocardial collagen type ?(Col ?),collagen type ?(Col ?)on the Ang ? induced rat cardiac fibroblasts(CFs)in order to explore the possible regulatory mechanisms of TRPM7/Smad2 signaling pathway in the remodeling of SAN and atrium in rats with SSS and collagen synthesis of CFs.Methods: 1.Histomorphology of SAN in normal SD rats were observed by HE staining after tissues in SAN region were sectioned continuously;the distribution of SAN and myocardial tissue collagen content in normal SD rats were observed by PASMMasson staining;HCN4 expression of SAN in normal SD rats were observed respectively by immunohistochemistry and immunofluorescence assay;It was indicated the expression of HCN4 stained as brown or green fluorescence was in the same site.2.20% sodium hydroxide infiltration was used to established SD rats SSS model by the operation method.Rats were divided into five groups: 1)normal control group(n = 8);2)sham group(n = 10);3)sick sinus syndrome(SSS)group was divided into three subgroups(SSS A group with 20~30% decrease of heart rate,n = 40;SSS B group with 31~40% decrease of heart rate,n = 40;SSS C group with 41~50% decrease of heart rate,n = 40).Postoperative SSS rats were divided into four observation points,which were the first,second,third,forth week after operation;SD rats SSS model's SAN histomorphology was observed by HE staining;The distribution of SAN and myocardial tissue collagen content in SD rats SSS model were observed by PASM-Masson staining;expression levels of HCN4,TRPM7,Smad2 in SD rats SSS model were observed by immunohistochemistry;ELISA method was employed to measure the level of Ang ?,Col ?,Col ? in serum and atrial tissue;Real-time PCR was used to measure the TRPM7 mRNA expression in SAN region tissue,and in the same region,TRPM7,Smad2 protein expression were measured by Western-blotting.3.The culture technique of tissue explants adherent dry method was applied to culture CFs in the SD rat's SAN region;CFs were identified by immunocytochemistry and immunofluorescence;the collagen level of supernatant was measured by ELISA;TRPM7 mRNA expression of CFs were tested by Real-time PCR;Western-blotting was used to measure TRPM7,Smad2 protein expression;TRPM7 expression was down-regulated by transfection of si RNA(small interfering RNA,si RNA)and overexpressed TRPM7 by transfection of plasmid DNA vector,for observing whether Smad2 expression mediated by Ang ? via TRPM7 pathway to influence on CFs' collagen synthesis.Results: 1.Anatomical location of SAN in SD rats: 1)SAN was crescent-shaped that located at the junction of superior vena cava and right atrial appendage and wall of superior vena cava of the beginning part of the border area,divided into three parts,including head,body and tail,the head is attached to the outer wall of the vena cava,the body was located at the junction of superior vena cava and right atrial and the tail attached to the outer wall of the right atrial.2.Histomorphological characteristics of SAN in SD rats: the P cells,T cells,ganglion cells and a small amount atrial myocytes are major population of SAN region.Head and tail are smaller looser,the body has larger size and much density,there are abundant reticular fibers in the interstitial tissue.The surface of SAN visible SAN artery is observed in the SAN surface,there are one to two branches in the SAN.SAN region in SD rat is surrounded by the surrounding loose connective tissue and adipose tissue,which there are clear boundaries between SAN and the superior vena cava and right atrium.P cell in SAN showed brown by HCN4 immunohistochemical staining and green fluorescent distributed in P cell membrane as punctate,circular or irregular short shaped by HCN4 immunofluorescence staining.3.Establishment of SSS model of SD rats: No death was seen in control and Sham groups,there was not significant difference in heart rate(P>0.05);Mortality of SSS C group was significantly higher than the SSS A group and SSS B group(P<0.05),there was not significant difference between SSS A group and SSS B group(P>0.05);heart rate of SSS A group gradually returned,almost recovered to preoperative heart rate at the forth week;the heart rate of SSS B group was stable after 2 to3 weeks of operation,and 31~40% lower than the preoperative heart rate;2)SAN rats HE staining showed: compared with the Control group,rats sinus SSS1 group tissue ganglion cell structure is more clear,a small amount of connective tissue nuclear proliferation and increased epithelial cell can be seen in ganglion gap;structures of SSS2 group,SSS3 group,SSS4 group sinus tissue ganglion cell were unclear,a large number of connective tissue proliferation was observed,vacuolation was also visible in SSS3,SSS4 group sinus tissue;Masson collagen staining showed: the growth of SSS group atrium collagen tissue had a time-dependent effect,SAN myocardial tissue collagen in SSS1 group were significantly increased compared with the Sham group(P<0.01),collagen of SSS3 group and SSS4 group further increased(P<0.05);there were not significant differences among control group,Sham group and SSS1 group on the HCN4 protein levels(P>0.05);HCN4 protein expression of SSS2,SSS3,SSS4 groups were significantly lower compared with Sham group(P<0.01).4.Animal experiments: 1)time-dependent effect was observed on Ang ? levels in serum of rat models and Ang ? and Col ?,Col ? levels in SAN region tissue in rat models,Ang ? levels in serum of rat models and Ang ? and Col ?,Col ? levels in SAN region tissue in rat models were significantly higher than the control group(P<0.05),three weeks after operation,Ang ? levels both in serum and in SAN region tissue of rat models reached the peak;There were no significant difference to be found between Sham and control groups(P>0.05);Col ?,Col ? levels in the serum of rat models showed there were no significant difference(P>0.05);2)immunohistochemistry study: time-dependent effect was observed on TRPM7,Smad2 expression in SAN and right atrial tissue of rat models,TRPM7,Smad2 expression of SSS1 rats were significantly increased compared to the control group(P<0.05),TRPM7,Smad2 expression of SSS2,SSS3,SSS4 rats were further increased(P<0.01);3)Real-time PCR: TRPM7 m RNA expression of SAN region tissue in SSS rats showed a time-dependent increasing,TRPM7 m RNA expression of SAN region tissue in SSS1 rats was significantly higher compared with the control group(P<0.01),TRPM7 m RNA expression in SSS2,SSS3,SSS4 rats significantly increased compared with the control group(P<0.01);Western-blotting: TRPM7 and Smad2 protein expression in SAN region of rat SSS models showed a time-dependent increasing,TRPM7 and Smad2 protein expression of SSS1 group were significantly higher compared with the control group(P<0.01),TRPM7 and Smad2 protein expression of SSS2,SSS3,SSS4 groups further increased compared with the control group(P<0.01),There were no significant difference to be found between Sham group and Control group(P>0.05).5.Cell Experiments: 1)Ang ? could promote CFs collagen synthesis in a dosedependent effect;2)losartan could inhibite CFs collagen synthesis in a dosedependent effect by Ang ?;3)Ang ? could promote TRPM7 and Smad2 m RNA and protein expression in CFs;4),TRPM7 gene expression reduced by transfection of TRPM7 specific si RNA,suppressing Ang ?-induced CFs Smad2 protein expression,reducing Ang ?-promoted CFs collagen synthesis;TRPM7 expression was upregulated by transfecting a plasmid DNA vector,enhancing the Ang ?-induced CFs cell Smad2 protein expression,strengthening Ang ?-induced CFs collagen synthesis.Conclusions: 1.Surgical method with usage of sodium hydroxide infiltration can establish a stable rat SSS model;2.Renin-angiotensin-aldosterone system(RAAS)of myocardial tissue in SAN region of SSS rat models can be activated,and promote myocardial fibrosis in SAN region tissue via TRPM7/Smad2 signaling pathway;3.losartan can inhibit Ang ?-induced collagen synthesis and expression of TRPM7/ Smad2 proteins;4.Activation of TRPM7 / Smad2 signaling pathway may be an important mechanism for both promoting myocardial fibrosis in SSS rats and Ang ?-induced CFs collagen synthesis.