Inhibition Of Farnesyl Pyrophosphate Synthase Improves Cardiac Remodeling Induced By Pressure Overload

Part 1 Inhibition of farnesyl pyrophosphate synthase improves chronic cardiac remodeling induced by pressure overloadBackground:Farnesyl pyrophosphate synthase(FPPS)is a vital branch-point enzyme in the mevalonate pathway.FPPS catalyzes isopentenyl pyrophosphate(IPP)and dimethylallyl pyrophosphate(DMAPP)to synthetise geranyl pyrophosphate(GPP)and further conjoins GPP with IPP to farnesyl pyrophosphate(FPP).FPP is the very substrate not only in cholesterol and coenzyme Q biosynthesis,but also in isoprenylation of small GTPases.Our previous studies have reported that inhibition of FPPS attenuates angiotensin ?-induced cardiac remodeling while the upregulation of Ras and FPPS were found in pressure overload rat model.In this study,we constructed an FPPS interfering mouse model.These mice were subjected to abdominal aortic constriction or sham operation to further investigate the effect and mechanism of FPPS in cardiac remodeling induced by pressure overload.Objective:To investigate the effect and mechanism of FPPS in cardiac remodeling induced by pressure overload.Methods:(1)Individual interfering sequences were designed to target to the gene of FPPS.The sequences were packaged by lentivirus.The interfering efficiency of each sequence in the infected neonatal cardiomyocytes was validated by western blot analysis.(2)The efficient lentivirus was microinjected through the zona pellucida into perivitelline space of zygotes of C57BL/6J mice.(3)Positive transgenic mice were screened and the interfering efficiency in the heart was validated by western blot analysis.The interfering efficiencies in their offsprings were aslo identified.(4)F3 generation,male FPPS interfering mice and their non-transgenic littermate control were subjected to abdominal aortic constriction or sham operation.(5)Tail blood pressures were measured and M-mode echocardiographic images were obtained 12 weeks after operation or sham operation.(6)The size of myocardial cells and the level of cardiac fibrosis were analyzed by histopathology.(7)The expressions of FPPS and its downstream proteins were measured by western blot analysis.(8)The activation of Ras,RhoA,Cdc42 and Racl was measured by G-LISA kit.(9)The expressions of some embryonic genes were measured by real-time PCR.Results:(1)The interfering sequence named pLVT202 can inhibit the expression of FPPS in myocardial cells.(2)FPPS interfering transgenic mice were successfully constructed.The expression of FPPS in the heart of transgenic mice was decreased about 50%compared with that of non-transgenic mice.The offsprings showed a similar interfering efficiency.(3)The heart of mice was shown significantly enlarged,myocardial hypertrophy and fibrosis 12 weeks after operation.(4)12 weeks after operation,FPPS Interfering could reduce the indexes of heart failure,decrease the level of myocardial hypertrophy and fibrosis and improve the ejection fraction induced by pressure overload.(5)12 weeks after operation,FPPS Interfering could decrease the activation of Ras protein and ERK1/2 phosphorylation induced by pressure overload,but could not decrease the elevated activated RhoA level.Conclusion:Interfering of FPPS could partly improve heart failure,myocardial hypertrophy and fibrosis induced by pressure overload.These effects may be attributed to the reduction of the activation Ras protein and its downstream ERK1/2 phosphorylation,which was related to the proliferation and fibrosis of myocardial cells.Part 2 Construction of cardiac conditional farnesyl pyrophosphate synthase knockout mouse modelBackground:Farnesyl pyrophosphate synthase(FPPS)is a a vital branch-point in the mevalonate pathway.FPPS catalyzes isopentenyl pyrophosphate(IPP)and dimethylallyl pyrophosphate(DMAPP)to synthetise geranyl pyrophosphate(GPP)and further conjoins GPP with IPP to farnesyl pyrophosphate(FPP).FPP is the very substrate not only in cholesterol and coenzyme Q biosynthesis,but also in isoprenylation of small GTPases.Knockout mouse model is a better model than RNA interfering model in the investigation of specified gene,but FPPS knockout model has not been reported.In order to further study the mechanism of FPPS in cardiac remodeling,we construct cardiac conditional FPPS knockout mouse model.Objective:To construct cardiac conditional farnesyl pyrophosphate synthase knockout mouse model.Methods:(1)Conditional knockout targeting FPPS vector was constructed.(2)The cko-targeting vector was linearized and electroporated into ES cells.The clones with right sequences were screened and implanted into the oviducts of pseudopregnant female mice.(3)The chimeric mice were identified and the FPPS-Flox homozygote mice were obtained.(4)FPPSfl/flCre+/-mice with myocardial specific expression of recombinant Cre were obtained.The efficiency of cardiac specific knockout FPPS was validated after tamoxifen administration.Results:(1)FPPS-Flox homozygous mice were successfully obtained.FPPSfl/fl nCre+/-mice with myocardial specific expression of recombinant Cre were obtained from FPPS-Flox homozygous mice.(2)The expression of FPPS in the heart of knockout mice was decreased about 80%compared with that of wild-type mice 8 weeks after tamoxifen administration.Conclusion:We constructed a mouse model that farnesyl pyrophosphate synthase was conditional knockout in the heart.