The Role Of Metabolic Remodeling In Cardiac Hypertrophy Promoted By UT-B Gene Knock Out In Mice

Background:Urea Transporter B（UT-B）expressed in multiple tissues such as kidney,heart, brain etc, is a membrane channel protein mediating the transmembranetransport of urea. In our previous work, we found that in UT-B knock-out mice, theserum and heart urea levels were increased; heart block occurred from16weeks old,and progressed to left ventricular remodeling, impaired cardiac function andInterstitial fibrosis in aging mice(52weeks). The results of left ventricular myocardialdifferential proteomics showed that UT-B knock-out mice have a higher level ofglycolysis and fatty acid β-oxidation related key enzyme’s expression. So we haveproposed the hypothesis that intracellular urea accumulation could lead to disorders ofmyocardial energy metabolism, promoting cardiac hypertrophy in UT-B knock-outmice. In the present study we observed the Morphological changes in UT-B knock-outmice under the condation of aging load (52weeks) and pressure load (4weeks afterabdominal aorta coarctation in16weeks mice), compared with wild type mice, toexplore the susceptibility of UT-B knockout mice to myocardial hypertrophy; Then,we analyzed the glucose oxidation, fatty acid oxidation, glycolysis level andmitochondrial function of myocardium in16and52weeks UT-B knock-out mice,compered with wild type mice, thereby verifing the hypothesis above, revealing therole of myocardial metabolic remodeling in the mechanisms of myocardialhypertrophy in UT-B knock-out mice, searching new targets for the prevention andtreatment of myocardial hypertrophy.1. Myocardial hypertrophy in UT-B knock-out mice is more easily to happenunder chronic stress. We have observed the morphological changes of52-week-old UT-B knock-outmice. The results demonstrated that heart weight and cell diameter were increased,interstitial fibrosis appeared, the phenomenon of myocardial hypertrophy wasobviously different. MDA level in their heart was increased, while the expression ofANP was elevated, electron microscopy results showed that there existed swelling ofmyocardial mitochondria, increased lipid droplets, which indicating that fatmetabolism disorders, and increased level of oxidative stress may take part in itspathogenesis.We performed an abdominal aortic constriction on16-week-old UT-B knock-outmice and wild-type mice. The results revealed that UT-B deficiency also acceleratedthe process of myocardial hypertrophy and myocardial remodeling, companying withchronic stress and ROS level of myocardial cells also increased significantly (p <0.05).2. The changes of glycometabolism, lipid metabolism, and mitochondrialfunction in cardiomyocytes of16-week-old UT-B knockout mice.We discovered that mRNA expression of the key enzymes in glycolytic isincreased through PCR array glucose metabolism chip detection of myocardial tissuein UT-B knock-out mice(p<0.05), the expression of ENOA, key enzyme of glycolysis,increased significantly (p<0.05); acidity of myocardial cells also increased markedly(p <0.05).Through western blot assay we found that ACAD(s), key enzyme of fattyacid oxidation, and PPARα, important regulatory factor expressed much less inmyocardial tissue of UT-B knock-out mice compared with those of wild type mice.The above conclusion indicated that unusual urea transportment in cardiomyocytes ofUT-B knock-out mice lead to glycometabolism abnormalities may be the primarycause for the increased susceptibility of myocardial hypertrophy.Analyzing protein expression discrepancy in myocardial mitochondrial of16-week-old UT-B knock-out mice using Nanoscale LC-MS/MS, we found that50kinds of proteins had obvious modulation, including mitochondrial electron transportchain (ETC) complex I, III, IV, and V relevant subunits, related enzymes of oxygen free radicals removing proteins, their expression levels were significantly decreasedwith age (p<0.05); activity of complex I and IV significantly decreased too (p<0.05),lower mitocondria related ATP levels appeared (p<0.01), mitochondrial mtDNAmutations occurred at many points. Detecting ROS and mitochondrial Δ Ψ m level ofmyocardial using flow cytometry in UT-B knock-out mice, we discovered that, ROS levelincreased obviously (p<0.01), Δ Ψ m reduced (p<0.05). The above results showed that thereexsits distinctly abnormal of mitochondrial function in UT-B knock-out mice. UsingMS analysis, we found the L-arg content in myocardium of16-week-old UT-Bknockout mice was significantly decreased (p<0.05); the NO content decreased(p<0.05), eNOS, Arg1and Ass1protein expression were significantly decreased(p<0.05), the results illustrate the urea cycle disorders in myocardium of16-week-oldUT-B knockout mice.3. The changes of glycometabolism, lipid metabolism, and mitochondrialfunction in cardiomyocytes of52-week-old UT-B knockout mice.Modulation of ETC complex relevant subunits and oxygen free radicalsremoving key enzymes occurred in16-week-old mice, continued to reduce in52-week-old UT-B knock-out mice (p<0.05) using Nanoscale LC-MS/MS analysis,key enzymes in glycometabolism, for instance, isocitrate dehydrogenase and succinatedehydrogenase had a lower expression level, while expression of ACAD and PPARαis obviously lower than the wild-type mice. The above conclusion indicated thatglucolipid metabolic abnormalities aggravated with age and may be the primary causeof increased oxidative stress and myocardial hypertrophy.In conclusion, urea transport defect in UT-B knock-out mice leads to urea cycledisorders, TCA block, oxidative phosphorylation impairment in mitochondrial,glucolipid metabolic abnormal, increased level of oxidative stress, finally results inmyocardial hypertrophy and myocardial remodeling.