Abstract 412: The Role of ETC Complexes I and II in the Respirasome Assembly in Heart Mitochondria

Abstract

Mitochondrial electron transport chain (ETC) plays an important role in ATP synthesis and its dysfunction is associated with human diseases. Recent cryo-electron microscopy studies revealed that individual ETC complexes are assembled into supercomplexes (SCs). Structural organization and physiological role as well as mechanisms of assembling and maintenance of the SCs are yet not completely understood. ETC complexes contribute unequally to structural organization of SCs, particularly the respirasome, the main SC which contains complexes I, III, and IV in various stoichiometries. However, many questions related to structural organization of the respirasome, particularly, a possible role of complexes I and II in respirasome formation remain unclear. Here, we focus on the role of complexes I and II in structural organization of SCs using both genetic and chemical inhibition of complex I and complex II in H9c2 cardiac myoblast cells and isolated cardiac mitochondria. Respiratory SCs were analyzed by blue native polyacrylamide gel electrophoresis. Pharmacological inhibition of complex I by rotenone and complex II by malonate stimulated dissociation of respirasome. Rotenone inhibited whereas malonate had no effect on calcium-induced mitochondrial swelling, a marker of permeability transition. On the other hand, the rate of mitochondrial ROS production was remarkably (>5 fold) stimulated by rotenone but not by malonate. Knockdown of the complex I subunit NDUFA11, which according to cryo-electron microscopy studies interacts with complex III in respirasome formation, stimulated dissociation of respirasome and reduced the activity of complexes I, III, and IV but not complex II. Downregulation of the SDHC subunit of complex II had no effect on the respirasome assembly but reduced the activity of complexes II and IV. In addition, knockdown of NDUFA11 or SDHC significantly reduced ATP levels in the cells. Overall, these studies for the first time provide biochemical evidence that NDUFA11 subunit of complex I is important for structural integrity of respirasome whereas complex II is less likely contribute to the integrity of respirasome.