Fig 1: Effects of Vigabatrin on apoptosis in cardiomyocytes. Cultured neonatal mouse cardiomyocytes were infected with an adenoviral vector expressing ABAT (Ad-ABAT) or GFP (Ad-GFP) and then exposed to palmitate or oleate (0.5 mM) in the presence of vehicle or Vigabatrin (500 µM), a selective inhibitor of ABAT for 48 h. (a) ABAT’s GABA catabolic activity. Data are mean ± SD, n = 5 in each group. * p < 0.05 vs. Ad-GFP + Vehicle, # p < 0.05 vs. Ad-ABAT + Vehicle. (b) Caspase-3 activity. Data are mean ± SD, n = 5 in each group. * p < 0.05 vs. Ad-GFP + Oleate + Vehicle, or Ad-GFP + Oleate + Vigabatrin, # p < 0.05 vs. Ad-GFP + Palmitate + Vehicle, or Ad-GFP + Palmitate + Vigabatrin. (c) DNA fragmentation. Data are mean ± SD, n = 5 in each group. * p < 0.05 vs. Ad-GFP + Oleate + Vehicle, or Ad-GFP + Oleate + Vigabatrin, # p < 0.05 vs. Ad-GFP + Palmitate + Vehicle, or Ad-GFP + Palmitate + Vigabatrin.
Fig 2: Effects of transgenic ABAT over-expression on mitochondrial function, oxidative stress and caspase-3 activation in HFD-fed mouse hearts. Transgenic mice with ABAT over-expression (TG) and their littermate controls (WT) were fed a ND (normal diet) or HFD (high fat diet) for 4 months. (a) Mitochondrial DNA (mtDNA) copy numbers in the heart. Data are mean± SD. n = 5–7 per group. * p < 0.05 vs. ND + WT, # p < 0.05 vs. HFD + WT. (b) ATP production in the heart. Data are mean± SD. n = 7–8 per group. * p < 0.05 vs. ND + WT, # p < 0.05 vs. HFD + WT. (c–e) Assessment of oxidative stress in the heart. Data are mean ± SD. n = 6–8 per group. * p < 0.05 vs. ND + WT, # p < 0.05 vs. HFD + WT. (c) ROS production, (d) MDA production and (e) protein carbonyl. (f) Apoptosis was assessed by caspase-3 activity. Data are mean± SD. n = 5–6 per group. * p < 0.05 vs. ND + WT, # p < 0.05 vs. HFD + WT.
Fig 3: Effects of ABAT over-expression in palmitate-incubated neonatal mouse cardiomyocytes. Cultured neonatal mouse cardiomyocytes were infected with an adenoviral vector expressing ABAT (Ad-ABAT) or beta-gal (Ad-gal) and then exposed to palmitate or oleate (0.5 mM for 48 h). (a) ABAT protein expression and activity in cardiomyocytes. Left panel: a representative western blot for ABAT and GAPDH. Right panel: ABAT activity. Data are mean ± SD. n = 5 per group. * p < 0.05 vs. Ad-gal. (b) Representative microphotographs of MitoSOXTM Red staining (red) and nucleus staining with Hoechst33324 (blue). (c) Quantification of MitoSOXTM Red staining intensity. Data are mean ± SD. n = 4 per group. * p < 0.05 vs. Oleate + Ad-gal. # p < 0.05 vs. Palmitate + Ad-gal. (d) Caspas-3 activity. Data are mean ± SD. n = 5 per group. * p < 0.05 vs. Oleate + Ad-gal. # p < 0.05 vs. Palmitate + Ad-gal. (e) DNA fragmentation. n = 5 per group. * p < 0.05 vs. Oleate + Ad-gal. # p < 0.05 vs. Palmitate + Ad-gal. (f) ATP production. n = 4 per group. * p < 0.05 vs. Oleate + Ad-gal. # p < 0.05 vs. Palmitate + Ad-gal.
Fig 4: HFD results in a reduction of ABAT in mouse hearts. After 4 months of high fat diet (HFD) feeding, cDNA microarray determined differential expression of genes in mouse hearts. (a) Heat map of differential gene expression. (b) The mRNA level of Abat relative to GAPDH was analyzed by RT-PCR. Data are mean ± SD, n = 5 per group. * p < 0.05 vs. ND (normal diet). (c) ABAT protein level was detected by western blot analysis. Upper panel: a representative western blot for ABAT and GAPDH from 4 out of 7 different hearts in each group. Bottom panel: quantification of ABAT/GAPDH ratio. Data are mean ± SD, n = 7 per group. * p < 0.05 vs. ND. (d) ABAT activity was measured by an enzymatic activity assay. Data are mean ± SD, n = 9–11 per group. * p < 0.05 vs. ND.
Fig 5: Western blots of catalase and metabolic proteins after 4-weeks of HFD(A) Catalase blot, loading control, and graph of quantification.(B) Blots of PDK4, HMGcs2, BDH1, ABAT, Ponceau loading control, and graph of quantifications adjusted for loading. Mean + SEM, * = p < 0.05 by * = p < 0.05, ** = p < 0.01, *** = p < 0.0001.
Supplier Page from Abcam for Anti-ABAT/GABA-T antibody [EPR4433]