Effect Of Mevalonate Pathway Enzymes On Myocardial Ischemia/Reperfusion Injury

Part 1 Knock-down of Farnesyl Pyrophosphate Synthase Protects Heart-derived H9c2 Cells against Hypoxia/Reoxygenation InjuryBackground:Myocardial infarction(MI)is the most serious manifestation of coronary heart disease(CHD).Timely recovery of the coronary flow by myocardial reperfusion is the most effective therapeutic measure.However,the process of myocardial reperfusion can induce further myocardial injury,a phenomenon known as myocardial ischemia reperfusion injury(MIR).Farnesyl pyrophosphate synthase(FPPS)is a key enzyme in the mevalonate pathway.It plays an important role in isoprenoid biosynthesis.It catalyzes isopentenyl pyrophosphate and geranyl pyrophosphate to form farnesyl pyrophosphate(FPP)and geranylgeranyl pyrophosphate(GGPP).Both FPP and GGPP are important in the activation of small GTPase protein such as Racl.Studies have incidated that FPPS has a significant effect on cardiac hypertrophy and cardiac remodeling.However,the role for FPPS in MIR injury is still not clear.Other studies have also revealed that the activation of Racl plays an important role in MIR.Aim:In this study,we investigate the effect of FPPS on MIR injury in H9c2 cells which were subjected to hypoxia/reoxygenation(HR)to mimic MIR.Methods:(1)Cultured cells were transfected with pE-mFPPS,shFPPS or pE-GFP.Expression levels of FPPS were measured by RT-PCR and western blot.(2)Transfection cells in plates were placed in a sealed chamber with anaerobic pouch at 37°C for 8 hours.Hypoxia was followed by 4 hours reoxygenation.(3)A CCK assay was used to assess cell viability.(4)Lactate dehydrogenase(LDH)or superoxide dismutase(SOD)was examined using a LDH Assay Kit and SOD Assay Kit.(5)Apoptotic cell death was measured using a PE AnnexinV Apoptosis Detection Kit.(6)Racl activity was determined by an absorbance-based G-LIS A Activation Assay Biochemistry Kit.(7)ROS production was measured using cellular ROS Red Fluorescence Assay Kit.Results:(1)FPPS expression levels were highly increased in pE-mFPPS group,FPPS expression levels were decreased in shFPPS group.(2)Overexpression of FPPS attenuated cell proliferation and FPPS silencing improved cell proliferation.(3)Overexpression of FPPS increased LDH and SOD levels and FPPS silencing decreased LDH and SOD levels.(4)Overexpression of FPPS increased HR-induced apoptosis and FPPS silencing decreased HR-induced apoptosis in H9c2 cells.(5)The inhibition of FPPS can decrease Racl activity.(6)Overexpression of FPPS increased ROS production and FPPS silencing decreased ROS production.Conclusion:The inhibition of FPPS can protect heart-derived H9c2 cells against hypoxia/reoxygenation injury,and overexpression of FPPS increased HR injury.The mechanisms of this phenomenon may be related to the alteration ofRacl activity.Part 2 Overexpression of farnesyl pyrophosphate synthase increases myocardial ischemia/reperfusion injury in miceBackground:Ischemic heart disease is a main cause of death and disability around the world.Timely recovery of the coronary flow by myocardial reperfusion is the most effective therapeutic measure.However,the process of myocardial reperfusion can induce further myocardial injury,a phenomenon known as myocardial ischemia reperfusion injury(MIR).Farnesyl pyrophosphate synthase(FPPS)is a key enzyme in the mevalonate pathway.It plays an important role in isoprenoid biosynthesis.It catalyzes isopentenyl pyrophosphate and geranyl pyrophosphate to form farnesyl pyrophosphate(FPP)and geranylgeranyl pyrophosphate(GGPP).Both FPP and GGPP are important in the activation of small GTPase protein such as Racl.The results in the first part of our study showed that overexpression of FPPS increased heart-derived H9c2 cells against hypoxia/reoxygenation injury.This phenomenon is associated with the increase of Racl activity,and subsequently improvement of ROS production.Aim:We investigate the effects of FPPS on myocardial ischemia reperfusion injury using transgenic(Tg)mice,and explore the relative mechanisms.Methods:Tg mice with overexpression of FPPS were used as an experimental model.(1)The expression levels of FPPS mRNA and protein were measured using real-time polymerase chain reaction(RT-PCR)and western blot,respectively.(2)Mice were treated with ischemia/reperfusion using retrograde Langendorff apparatus.Heart functions including Left ventricular developed pressure(LVDP),LVEDP,maximum rate of intraventricular pressure development(dp/dtmax)and relaxation(-dp/dtmax),and heart rate were measured before and post-ischemia.(3)The cumulative amounts of creatine kinase(CK)or lactate dehydrogenase(LDH)released in the coronary effluent was measured using CK Assay Kit or LDH Assay Kit according to manufacturer's protocols.(4)Infarct size was measured using triphenyltetrazolium chloride(TTC).(5)Racl activity was determined by an absorbance-based G-LISA Activation Assay Biochemistry Kit.(6)ROS generation was measured using Tissue ROS Fluorescence Assay Kit.Results:(1)FPPS expression levels were highly increased in Tg mice.(2)Overexpression of FPPS decreased the recovery of LVDP,LVEDP,dp/dtmax and-dp/dtmax in isolated IR mouse hearts.The heart rate and coronary flow rate had no significant difference among all groups.(3)Overexpression of FPPS increased CK and LDH levels in coronary effluent.(4)Overexpression of FPPS aggravated myocardial infarction in isolated IR mouse hearts.(5)Overexpression of FPPS increased the activation of Racl in isolated IR mouse hearts.(6)Over expression of FPPS increased ROS production in isolated IR mouse hearts.Conclusion:Overexpression of FPPS increased myocardial ischemia reperfusion injury in mice,and this phenomenon is associated with the increase of Racl activity,and subsequently improvement of ROS production.Part 3 Effect of Geranylgeranyl Pyrophosphate Synthase on Hypoxia/Reoxygenation-Induced Injury in Heart-derived H9c2 CellsBackground:Timely recovery of the coronary flow by myocardial reperfusion is the most effective therapeutic measure of ischemic heart disease.However,the process of myocardial reperfusion can induce further myocardial injury,a phenomenon known as myocardial ischemia reperfusion injury(MIR).GGPPS catalyzes the synthesis of GGPP,which plays an important role in the activation of small GTPase protein such as Racl · The results of the first two parts of our study showed that the alteration of FPPS expression plays an important role in MIR,and this phenomeinoin is associated with the increase of Racl activity,and subsequently improvement of ROS production.Aim:In this study,we investigate the effect of GGPPS on MIR injury in H9c2 cells which were subjected to hypoxia/reoxygenation(HR)to mimic MIR.Methods:(1)Plasmids include pAV-rGGPPS,shGGPPS and shGFP were constructed.(2)Cultured cells were transfected with pAV-rGGPPS,shGGPPS and shGFP.Expression levels of GGPPS were measured by RT-PCR and western blot.(3)Transfection cells in plates were placed in a sealed chamber with anaerobic pouch at 37? for 8 hours.Hypoxia was followed by 4 hours reoxygenation.(4)A CCK assay was used to assess cell viability.(5)Lactate dehydrogenase(LDH)or superoxide dismutase(SOD)was examined using a LDH Assay Kit and SOD Assay Kit.(6)Apoptotic cell death was measured using a PE AnnexinV Apoptosis Detection Kit.(7)Racl activity was determined by an absorbance-based G-LIS A Activation Assay Biochemistry Kit.(8)ROS production was measured using cellular ROS Red Fluorescence Assay Kit.Results:(1)GGPPS expression levels were highly increased in PAV-rGGPPS group,GGPPS expression levels were decreased in shGGPPS group.(2)Overexpression of GGPPS attenuated cell proliferation and GGPPS silencing improved cell proliferation.(3)Overexpression of GGPPS increased LDH and SOD levels and GGPPS silencing decreased LDH and SOD levels.(4)Overexpression of GGPPS increased HR-induced apoptosis and GGPPS silencing decreased HR-induced apoptosis in H9c2 cells.(5)Overexpression of GGPPS increased Racl activity,GGPPS silencing decreased Rac1 activity.(6)Overexpression of GGPPS increased ROS production and GGPPS silencing decreased ROS production.Conclusion:Overexpression of GGPPS aggravates HR-induced injury in heart-derived H9c2 cells,and GGPPS silencing protects heart-derived H9c2 cells against HR-induced injury.The mechanisms of this phenomenon may be related to the alteration of Rac1 activity.