Fig 1: Proteomic analysis of protein abundance in WT vs.Alkbh7-/-.Hearts from young male WT or Alkbh7-/- mice were analyzed by tandem mass tag LC-MS/MS as per the methods. (A) Volcano plot showing relative levels of 3737 proteins. X-axis shows Log10 of fold change (Alkbh7-/- / WT) and Y-axis shows Log10 of significance (paired t-test, N = 5). Proteins crossing thresholds (gray lines) in upper left or right quadrants are labeled. (B) Activity of GLO-1 in WT or Alkbh7-/- heart cytosol. (C) Activity of GLO-2 in WT or Alkbh7-/- heart cytosol. (D) Western blot showing abundance of GLO-1 in WT or Alkbh7-/- heart cytosol, with Ponceau stained membrane below. (E) Quantitation of GLO-1 blot, normalized to protein loading. Bar graphs in panels B/C/E show means ± SE, N = 4–5, with p-values (paired t-test) shown above error bars. (F) Schematic showing the methylglyoxal detoxification system and its relationship to glycolysis. Abbreviations: AGEs: Advanced glycation end products, GSH: glutathione. MGO: methylglyoxal. In bar graphs, N for each group is shown in parentheses.
Fig 2: Blockade of cardioprotection in Alkbh7-/- by GLO-1 inhibition.Hearts from young male WT and Alkbh7-/- mice were Langendorff perfused and subjected to IR injury as in Figure 4, with delivery of 1 µM SBB-GSH-CpE for 10 min. prior to ischemia. (A) Cardiac function assessed by left ventricular balloon pressure transducer. Graph shows the product of heart rate multiplied by left ventricular developed pressure, as a percentage of the initial value. A significant drop in cardiac function was observed upon drug infusion in Alkbh7-/- only (see arrow and p-value). (B) Post IR staining with TTC for quantitation of myocardial infarct size. Representative TTC-stained heart slices are shown, with pseudo-colored mask images used for quantitation by planimetry (red = live tissue, green = infarct). Data are quantified below, with individual data points to shown N, and means ± SE. p-values (paired t-test) are shown above error bars. (C) Western blot showing abundance of DJ-1 in Alkbh7-/- and WT heart mitochondria. Ponceau stained membrane and quantitation are shown below. Bar graph shows means ± SE, N = 4, with p-values (paired t-test) shown above error bars. (D) Schematic showing proposed events that connect loss of ALKBH7 to cardioprotection. Via mechanisms that may include downregulation of Perilipin five and F-1,6-BPase 2, loss of ALKBH7 causes a shift in metabolism away from fatty acid oxidation (FAO) toward elevated glycolysis. GLO-1 is also upregulated, possibly in response to MGO elevation. Both elevated glycolysis and GLO-1 then protect against IR injury. The potential role of GLO-1 as a regulator of the metabolic shift toward glycolysis is also shown.
Fig 3: Protein immunoblot analysis. Immunoblotting of the proteins involved in mitochondrial and metabolic functions was performed in the hippocampi from the control, depression-susceptible, anxiety-susceptible, and insusceptible groups.Por, Idh2, Esd, Glo1, G6pdx, Aldh2, Dld, Dlat, Ogdhl, Anxal, Tpp2, Sdha, Prnp, Pkm, and Prdx6 were detected with their respective antibodies. Each blot corresponded to the five rats used in the analysis, and Coomassie blue staining was used as the loading control. Dep-Sus depression-Susceptible, Anx-Sus Anxiety-susceptible, Insus Insusceptible, Cont Control; n = 5, *p < 0.05, **p < 0.01
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