Deletion of Von Willebrand A Domain Containing Protein (VWA8) raises activity of mitochondrial electron transport chain complexes in hepatocytes

Abstract

VWA8 (Von Willebrand A Domain Containing Protein 8) is a AAA+ ATPase that is localized to the mitochondrial matrix and is widely expressed in highly energetic tissues. Originally found to be higher in abundance in livers of mice fed a high fat diet, deletion of the VWA8 gene in differentiated mouse AML12 hepatocytes unexpectedly produced a phenotype of higher mitochondrial and nonmitochondrial oxidative metabolism, higher ROS (reactive oxygen species) production mainly from NADPH oxidases, and increased HNF4a expression. The purposes of this study were first, to determine whether higher mitochondrial oxidative capacity in VWA8 null hepatocytes is the product of higher capacity in all aspects of the electron transport chain and oxidative phosphorylation, and second, the density of cristae in mitochondria and mitochondrial content was measured to determine if higher mitochondrial oxidative capacity is accompanied by greater cristae area and mitochondrial abundance. Electron transport chain complexes I, II, III, and IV activities all were higher in hepatocytes in which the VWA8 gene had been deleted using CRISPR/Cas9. A comparison of abundance of proteins in electron transport chain complexes I, III and ATP synthase previously determined using an unbiased proteomics approach in hepatocytes in which VWA8 had been deleted showed agreement with the activity assays. Mitochondrial cristae, the site where electron transport chain complexes are located, were quantified using electron microscopy and stereology. Cristae density, per mitochondrial area, was almost two-fold higher in the VWA8 null cells (P < 0.01), and mitochondrial area was two-fold higher in the VWA8 null cells (P < 0.05). The results of this study allow us to conclude that despite sustained, higher ROS production in VWA8 null cells, a global mitochondrial compensatory response was maintained, resulting in overall higher mitochondrial oxidative capacity.

Keywords: ADP, adenine dinucleotide phosphate; ANT, adenine nucleotide translocase; ATP, adenine trinucleotide phosphate; ETC, electron transport chain; Electron transport chain; HNF4, hepatocyte nuclear factor 4; Hepatocytes; Mitochondria; NADPH, nicotinamide adenine dinucleotide phosphate; OCR, oxygen consumption rate; PFO, perfringolysin; ROS, reactive oxygen species; TMHQ, Tetramethylhydroquinone; TMPD, N,N,N′,N'-Tetramethyl-p-phenylenediamine; VWA8; VWA8, Von Willebrand Domain-containing Protein 8.