The Effects Of UCP2 On Energy Metabolic Pathways In Septic Myocardium And The Underlying Mechanism

ObjectivesClinical study:To clarify the serum levels of pyruvate dehydrogenase kinases 1(PDK1)and lactate dehydrogenase(LDH)in septic patients,and to further evaluate the clinical value of PDK1/LDH in patients with sepsis.In-vitro experiments:We will firstly investigate the effect of uncoupling protein 2(UCP2)on the regulation of oxidative phosphorylation(OXPHOS)and glycolysis in cardiomyocytes during sepsis in an in vitro model of sepsis and explore the underlying mechanisms,with the aim of identifying new roles for UCP2 in the regulation of energy in sepsis.Secondly,we will investigate the specific significance of changes in pyruvate dehydrogenase(PDH)activity,PDK and LDH on the energy metabolic pathways in cardiomyocytes for finding evidences of PDH activity as a therapeutic target for sepsis.In-vivo experiments:To verify that UCP2 regulates pyruvate metabolic balance and ATP levels in mice myocardial tissue through PDH activity,PDK and LDH,and to investigate the effects of UCP2 on myocardial injury and cardiac function in UCP2-KO mice and UCP2-UP mice.MethodsClinical study:We collected serum from 167 ICU patients within 24 hours of admission for a single-center prospective observational study,including 90 patients in the sepsis group,37 in the septic shock group,and 40 in the ICU control group.The serum levels of PDK1 and LDH were tested by ELISA kit.All the necessary clinical data about patients were collected,and we used SPSS 26.0 and GraphPad Prism version 8.0 for statistical analysis and plotting.In-vitro experiments:The si-UCP2 and OE-UCP2 cardiomyocytes were constructed by transfecting AC 16 cardiomyocyte cells with lentivirus and adenovirus,respectively.In vitro sepsis model was constructed using LPS.At 6h,18h,and 48h,the kit measures cardiomyocyte glucose uptake,pyruvate,acetyl-CoA,and lactate levels.WB technology detects the expression levels of UCP2 and associated metabolic enzymes(PDH,LDH).OCR and ECAR were also detected using seahorse technology to assess cellular OXPHOS and glycolysis capacity.In-vivo experiments:We established Control group,Sham group and CLP group in WT mice,UCP2-KO mice,and mice with up-regulated UCP2 expression(UP-UCP2),respectively.Heart tissue and serum were taken after cardiac ultrasound assessment at 18h.Pathological staining and serum cTnI were used to assess the extent of myocardial injury and each group was tested separately for relevant protein expression levels(UCP2,PDK,PDH,etc.),inflammatory factor levels(TNF-α,IL-6),ATP levels,PDH and LDH activity with kits,and for relevant metabolites(pyruvate,lactate).ResultsClinical study:Serum PDK1 and LDH levels as individual biomarkers did not show good clinical value in both the Sepsis group and Septic shock groups;The levels of PDK1/LDH in the Septic shock group was statistically different between both the sepsis group(p=0.044)and ICU control group(p=0.011),and had good correlation with ScvO2(Spearman r2=0.42,p=0.009)and lactate(Spearman r2=-0.43,p=0.008).In predicting 28-day mortality in patients with sepsis,the best AUC was observed for PDK1/LDH(AUC,0.713;95%CI,0.589-0.836,p=0.002),and was higher than the AUC for PDK1,lactate,SOFA,and LDH.Additionally,patients with lower PDK1/LDH had higher 28-day mortality(log-rank p<0.05).The multivariate Cox proportional hazards model revealed that PDK1/LDH<0.1808 was the independent risk factors for 28-daymortality in sepsis.In-vitro experiments:UCP2 expression was significantly increased in LPS-stimulated cardiomyocytes.Compared to the control group,the LPS group showed decreased total ATP level,enhanced glycolysis but reduced OXPHOS capacity.At the same time,the levels of glucose uptake,pyruvate and lactate were significantly increased but acetyl coenzyme A decreased in cardiomyocytes after LPS.LPS also resulted in decreased PDH activity,increased PDK protein and PDH phosphorylation and enhanced LDH activity in cardiomyocytes.Glucose uptake was significantly increased in the OE-UCP2 group and promoted glycolytic ATP synthesis at 6h and 18h.We also found that changes in PDH activity had no effect on OXPHOS-ATP in cardiomyocytes after LPS stimulation.Changes in UCP2 expression also had no effect on OXPHOS-ATP at 6h and 18h.Overexpression of UCP2 increased PDH activity and preserved the reserve capacity of mitochondrial OXPHOS and increased the level of ATP synthesized by OXPHOS at 48h.In-vivo experiments:CLP caused decreased cardiac contractile function,myocardial tissue damage and decreased ATP levels in WT mice,UCP2-KO mice and UCP2-UP mice.PDH activity in myocardial tissue was significantly higher in the up-regulated UCP2 group than in the control group,while LDH activity was lower,which significantly balanced pyruvate metabolism in the myocardium of septic mice.Meanwhile,UCP2 increased ATP levels in septic cardiac tissue,and effectively protected cardiac contractile function and reduced the extent of myocardial injury in septic mice.Conclusion1.The decreased glucose uptake by cardiomyocytes during sepsis resulted in decreased glycolytic ATP production;2.Changes in PDH activity in septic cardiomyocytes cannot directly affect ATP synthesis by OXPHOS;3.In septic cardiomyocytes,protection of PDH activity and inhibition of LDH and PDK expression contribute more to the preservation of mitochondrial OXPHOS reserve capacity,which in turn increases OXPHOS-ATP synthesis in late sepsis;4.Overexpressed UCP2 increased glucose uptake by septic cardiomyocytes to promote glycolysis-ATP synthesis in the early stages of sepsis,while enhancing mitochondrial OXPHOS reserve capacity to contribute to an increase in 48h OXPHOS-ATP;5.UCP2 regulates the metabolic balance of pyruvate through active PDH and LDH and reduces metabolic disturbances in the myocardium of septic mice;6.PDK1/LDH is an independent influence factor on 28-day mortality in patients with sepsis;7.UCP2 may be a potential therapeutic target for reducing cardiomyocyte damage and protecting cardiac function in sepsis.