The Regulatory Effect Of Sphingosine-1-phosphate On Cardiomyoctye Hypertrophy

Objective:To explore the regulation of exogenous and endogenous sphingosine-1-phosphate?S1P?on cardiac hypertrophy and its possible mechanisms.Methods:1.The regulation of exogenous S1P on cardiac hypertrophy?1?Extraction and culture of primary cardiomyocytesThe primary cardiomyocytes were extracted with trypsin and type II collagenase digestion method and differential adherent separation method.Cardiomyocytes were cultured with complete medium of DMEM containing 10%FBS and 1%double antibody.?2?Exogenously administered S1P treatment with different concentration gradientsAfter neonatal cardiomyocytes were cultured for 48 hours,the serum-free DMEM medium was starved for 24 hours.And then different concentrations of S1P were added for another 24 hours.The experimental grouping and treatment were as follows:1)Normal control group?C group?:After 24 hours of starvation treatment,continue to culture for 24 hours using DMEM medium.2)S1P100nM group?S1P100 group?:after starvation treatment for 24h,continue to culture for 24h with DMEM medium containing S1P final concentration of 100nM.3)S1P1?M group?S1P1 group?:after starvation treatment for 24h,continue to culture for 24h with DMEM medium containing S1P final concentration of 1?M.4)S1P10?M group?S1P10 group?:after starvation treatment for 24h,continue to culture for 24h with DMEM medium containing S1P final concentration of 10?M.?3?Determination of ANP,BNP and MYH7 mRNA expression by RT-qPCRThe total RNA of primary cardiomyocytes of each group was extracted and cDNA was synthesized by reverse transcription method.RT-qPCR amplification reaction was used to measure the expression of ANP,BNP and MYH7 mRNA.?4?Western Blot was used to detect the relative expression levels of ANP,BNP and MYH7 proteins in cardiac hypertrophy?5?Observation of myocardial surface area changes in primary cardiomyocytes of neonatal rats by immunofluorescence labeling2.Correlation between exogenous S1P promoting cardiac hypertrophy and receptor subtypes?1?Experimental grouping and different drug treatmentThe isolated primary cardiomyocytes were cultured for 48 hours and randomly divided into eight groups,each of which was treated as follows:1)Normal control group?C group?:Primary cardiomyocytes were cultured for48 hours,starved for 24 hours,and cultured in DMEM medium for 24 hours.2)S1P1?M treatment group?S1P group?:primary cardiomyocytes were cultured for 48 hours,starved for 24 hours,and cultured for 24 hours in DMEM medium containing S1P final concentration of 1?M.3)S1PR₁ blocker W146 group?W146 group?:primary cardiomyocytes were cultured for 48h,starved for 24h,and cultured in DMEM medium containing W146with final concentration of 10?M for 24h.4)S1PR₂ blocker JTE013 group?JTE013 group?:primary cardiomyocytes were cultured for 48 hours,starved for 24 hours,and cultured for 24 hours in DMEM medium containing JTE013 with a final concentration of 10?M.5)S1PR₃ blocker CAY10444 group?CAY10444 group?:primary cardiomyocytes were cultured for 48h,starved for 24h,and continued to culture for24h in DMEM medium containing CAY10444 with final concentration of 10?M.6)S1P+W146 group:primary cardiomyocytes were cultured for 48 hours,starved for 24 hours,and cultured for 24 hours in DMEM medium with final concentration of S1P of 1?M and final concentration of W146 of 10?M.7)S1P+JTE013 group:primary cardiomyocytes were cultured for 48 hours,and starved for 24 hours,and cultured for 24 hours with DMEM medium containing S1P with a final concentration of 1?M and a final concentration of JTE013 of 10?M.8)S1P+CAY10444 group:primary cardiomyocytes were cultured for 48 hours,starved for 24 hours,and cultured for 24 hours in DMEM medium with final concentration of S1P of 1?M and final concentration of CAY10444 of 10?M.?2?Western Blot was used to detect the relative expression levels of ANP,BNP and MYH7 proteins in cardiac hypertrophy?3?Observation of myocardial surface area changes in primary cardiomyocytes of neonatal rats by immunofluorescence labeling3.Regulation of endogenous S1P on cardiac hypertrophy?1?Establishment of AngII-induced cardiac hypertrophy model in primary cardiomyocytes of neonatal ratsThe neonatal cardiomyocytes were cultured for 48 hours.They were treated with serum-free DMEM medium for 24 hours,and then treated with 100nM and 1?M AngII for 24 hours.RT-qPCR method was used to determine ANP,BNP and MYH7mRNA expression,Western Blot detection of ANP,BNP and MYH7 protein relative expression level,immunofluorescence labeling method to observe myocardial surface area changes in neonatal rat cardiomyocytes.?2?Construction of SK1,SK2 overexpressing lentiviral vector to observe the effect of increased endogenous S1P production on cardiac hypertrophyConstruction and packaging of SK1,SK2 overexpressed lentiviral vector and quality testing,used to infect primary cardiomyocytes,determine MOI and optimal infection conditions,and divide primary mouse cardiomyocytes into CON335 empty virus control Group?CON335 group?,CON335 empty virus control+AngII 1?M?CON335+AngII group?,SK1 overexpression lentivirus group?SK1 group?,SK1overexpression lentivirus+AngII 1?M?SK1+AngII group?,CON294 empty virus control Group?CON294 group?,CON294 empty virus control+AngII 1?M group?CON294+AngII group?,SK2 overexpression lentivirus group?SK2 group?,SK2overexpression lentivirus+AngII 1?M group?SK2+AngII group?,RT-qPCR was used to detect the expression of ANP,BNP and MYH7 mRNA in SK1 and SK2overexpressing lentiviruses.The relative expression of ANP,BNP and MYH7proteins in each group was determined by Western Blot.immunofluorescence method was used to observe myocardial cell surface area changes after SK1,SK2overexpression.?3?Construction of SK1,SK2 knockdown plasmid vector to observe the effect of reduce endogenous S1P production on cardiac hypertrophyThe SK1,SK2 knockdown plasmid vector was constructed,and the primary cardiomyocytes of SD rats were infected.The primary cardiomyocytes were divided into eight groups,namely SK1 knockdown plasmid control group?C1 group?,SK1knocking negative plasmid control+AngII 1?M group?C1+AngII group?,SK1knockdown plasmid group?Si-SK1 group?,SK1 knockdown plasmid+AngII 1?M group?Si-SK1+AngII group?,SK2 knockdown plasmid control group?C2 group?),SK2 knockdown plasmid control+AngII 1?M group?C2+AngII group?,SK2knockdown plasmid group?Si-SK2 group?,SK2 knockdown plasmid+AngII 1?M group?Si-SK2+AngII group?,RT-qPCR was used to determine the expression levels of ANP,BNP and MYH7 mRNA in SK1 and SK2 knockdown plasmids.The relative expression levels of ANP,BNP and MYH7 proteins in SK1 and SK2 knockdown plasmids were detected by Western Blot.Immunofluorescence was used to observe the changes of surface area of myocardial cells in each group after SK1 and SK2knockdown plasmid transfection.Results:1.The regulation of exogenous S1P on cardiac hypertrophyCompared with group C,ANP,BNP,MYH7 mRNA expression,protein relative expression level and myocardial cell surface area of S1P100 group,S1P1 group,S1P10 group increased significantly;the S1P1 group increased most significantly.2.Correlation between exogenous S1P promoting cardiac hypertrophy and receptor subtypesCompared with the S1P group,the relative expression levels of ANP,BNP and MYH7 proteins and the surface area of myocardial cells in S1P+W146 group and S1P+JTE013 group were significantly decreased,while this in S1P+CAY10444group were no significant change.3.Regulation of endogenous S1P on cardiac hypertrophy?1?Establishment of AngII-induced cardiac hypertrophy model in primary cardiomyocytes of neonatal ratsCompared with group C,ANP,BNP,MYH7 mRNA expression,protein relative expression level and myocardial cell surface area increased significantly in AngII100nM group,AngII 1?M group;Compared with AngII 100nM group,the outcome of AngII 1?M group was more significant.?2?Construction of SK1,SK2 overexpressing lentiviral vector to observe the effect of overexpression SK on cardiac hypertrophyCompared with the CON335 group,the ANP,BNP,MYH7 mRNA expression levels,the relative expression proteins and the surface area of myocardial cells increased significantly in the CON335+AngII group,while the above indicators in the SK1 group did not change significantly;compared with the CON335+AngII group,the expression levels of ANP,BNP and MYH7 mRNA,the relative expression proteins and the surface area of myocardial cells in SK1+AngII group were significantly decreased,which suggested that overexpression of SK1 can inhibit AngII-induced cardiac hypertrophy.The expression of ANP,BNP and MYH7 mRNA,the relative expression proteins and the surface area of myocardial cells in CON294+AngII group were significantly increased comparing with CON294 group,but there was no significant change in the SK2 group.Compared with the CON294+AngII group,there was no significant change in ANP and BNP mRNA expression,protein relative expression level and myocardial cell surface area showed no significant changes in SK2+AngII group.?3?Construction of SK1,SK2 knockdown plasmid vector to observe the effect of knockdown SK on cardiac hypertrophyCompared with C1 group,ANP,BNP and MYH7 mRNA expression levels,proteins relative expression levels and myocardial cell surface area increased significantly in C1+AngII group,while Si-SK1 group had no significant change;Compared with the C1+AngII group,the expression levels of ANP,BNP and MYH7mRNA,the relative expression proteins and the surface area of myocardial cells increased significantly in Si-SK1+AngII group.Compared with C2 group,ANP,BNP and MYH7 mRNA,the expression levels and the surface area of myocardial cells in C2+AngII group were significantly increased,while the Si-SK2 group had no significant change.Compared with C2+AngII group,ANP and BNP mRNA expression,the relative expression proteins and the surface area of myocardial cells in Si-SK2+AngII group were not significantly changed.Conclusion:1.Exogenous S1P has the effect of promoting cardiac hypertrophy.2.The role of exogenous S1P in promoting cardiac hypertrophy may be related to the activation of S1PR₁ and S1PR₂ receptors.3.The endogenous S1P induced by SK1 can inhibit the development of cardiac hypertrophy.4.The endogenous S1P induced by SK2 has no effect on the development of cardiac hypertrophy.