Fig 1: Prevention of Erk5 degradation rescued mitochondrial functions in ARCMs. a Prevention of Erk5 reduction by calpain-1 knockdown or MDL-28170 (30 µM for 16 h) was able to retrieve the mRNA level of key mitochondrial genes reduced by 8 h PA treatment. * or # P < 0.05, vs. PA-treated group. b Mitochondrial oxidation activities of ß-hydroxylacyl CoA dehydrogenase and pyruvate dehydrogenase (Pdh) were restored when calpain-1 was blocked. c FFA-induced oxidative damage was ameliorated followed by calpain-1 knockdown or inhibition, indicated by decreased protein carbonyl amount or active caspase-3 expression, respectively. n = 6 independent experiments per group. Actin is the protein loading control. Data are presented as means ± SD
Fig 2: Loss of Erk5 caused cellular injury in lipids overloaded hearts. a FFA extracted from heart tissues, and b Heart sections stained with Oil Red O, indicated that neutral lipids were increased in HFD-CKO hearts (scale bar: 20 µm). c Measurement of DAG content demonstrated an accumulation of its level in HFD-CKO hearts. d Triple quadrupole mass spectrometer analysis of ceramide species showed an altered ceramide species profile in CKO hearts. # P < 0.05 vs. chow-Flox, § P < 0.05 vs. HFD-Flox, *P < 0.05 vs. chow-CKO, n = 7 mice per group. e DHE staining determined more superoxide generation in HFD-CKO hearts (left panel, scale bar: 20 µm). Fluorescence intensity was quantified (right panel). f TUNEL assay by triple staining with DAPI (blue), anti-a-actinin antibody (red), and TUNEL (green) detected apparent apoptosis in HFD-CKO hearts (left panel, scale bar: 20 µm), arrows indicated TUNEL positive nuclei. The quantification of TUNEL positive nuclei is shown in bar graphs (right panel), n = 6 mice per group. g Immunoblot analysis showed increased active caspase-3 in the hearts of HFD-CKO mice. The quantifications are represented by the bar graphs. h Immunoblot analysis showed decreased Irs1 in HFD-CKO hearts. The quantification is represented by the bar graphs, n = 6 mice. Actin is the protein loading control. Data are presented as means ± SD
Fig 3: Schematic model of Erk5 protection against metabolic stress. FFA induces escalated ROS production from Gp91phox, which activates caplain-1, leading to a breakdown of Erk5. Loss of Erk5 and resultant Pgc-1a downregulation impose detrimental impacts on mitochondrial functions through accumulation of toxic lipids and vicious cycle of ROS. Prevention of Erk5 degradation or Erk5 restoration by AAV9 approach revives mitochondrial functions
Fig 4: Erk5 was required for mitochondrial function-related gene expression programmes. a Quantitative real-time PCR (qPCR) showed a decreased expression of Ppargc1a, Ppargc1b, Ppara, and Nrf1 in the hearts of HFD-CKO mice. The mRNA level of genes involved in FAO b, glycolytic action c, or mitochondrial biogenesis and OXPHOS d were assayed by qPCR. e The mRNA level of antioxidants was decreased in HFD-CKO hearts. Data are plotted as means ± SD (*P < 0.05, vs. HFD group, n = 7 mice per group)
Fig 5: Decreased expression and phosphorylation of Erk5 in the obesity/diabetic hearts. a Immunoblot analyses (antibody recognizing C-terminus of Erk5) showed a decrease of Erk5 expression in the hearts from C57BL/6J mice with 25-week HFD, db/db mice, ob/ob mice, or rhesus monkeys with metabolic syndrome (MS). Quantification of Erk5/Actin ratio is presented in the bar graph. Data are means ± SD (*P < 0.05, vs. control groups, n = 6 animals per group). b Immunoblot analyses substantiated that expression level of Erk5 (antibody recognizing N-terminus of Erk5), Mef2a and Mef2d was decreased in the obesity/diabetic hearts. c The phosphorylation levels of Erk5, Erk1/2, Jnk, p38, Ampk, Creb, and Mek5 in the hearts from mice with 16-week or 25-week HFD were examined by immunoblot analyses. Actin is the protein loading control
Supplier Page from Abcam for Recombinant human ERK5 protein