Abstract 854: The Role of Mitochondrial Permeability Transition in the Regulation of ETC Supercomplexes Assembly by OPA1

Abstract

Optic atrophy type 1 (OPA1), a dynamin-related GTPase is the inner mitochondrial membrane (IMM) protein, which, in addition to mitochondrial fusion, participates in maintaining the structural integrity of the IMM. The function and topography of the IMM is also regulated by changes in the matrix volume. Opening of the mitochondrial permeability transition pores (PTP) is the leading cause of mitochondrial matrix swelling. However, the cause-and-effect relationship between OPA1 proteolytic cleavage and PTP induction remains unknown. Here, we elucidated the role of PTP in OPA1-mediated regulation of electron transport chain (ETC) supercomplexes (SCs) assembly. First, intact mitochondria isolated from the rat heart were used to analyze the effect of PTP induction on the cleavage of long (L)-OPA1. Calcium-induced mitochondrial swelling increased L-OPA1 cleavage (66%, P<0.001) associated with accumulation of short (S)-OPA1. Inhibition of swelling by sanglifehrin A (a PTP inhibitor) prevented L-OPA cleavage whereas tert-butyl hydroperoxide (TBH) had no effect on L-OPA levels. Second, OPA1 knockdown (KD) by siRNA in HEK cells resulted in a 74% decrease (P<0.001) in OPA1 protein expression but did not change the proportion of L-OPA1 to total OPA. Mitochondria isolated from OPA1 KD cells showed reduced calcium-stimulated swelling with no changes in protein levels of cyclophilin D, a major PTP regulator. OPA1 KD cells demonstrated a 20% increase (P<0.05) in calcium-induced ROS generation. Analysis of individual ETC complexes revealed significant reduction of protein levels of the complexes I, III, and IV in OPA1 KD cells. Likewise, activities of complexes I and IV were reduced (48% and 37%, respectively, P<0.01 for both). OPA1 KD cells contained 38% (P<0.05) less ATP whereas the citrate synthase activity reflecting mitochondria mass was not affected. Blue native PAGE analysis demonstrated a 35% decrease (P<0.01) of high molecular weight proteins (>100 kDa) and a 24% decrease (P<0.05) of the respirasome, the main SC, levels in OPA1 KD mitochondria. We conclude that mitochondrial swelling by PTP opening stimulates OPA1 cleavage, and reduced OPA1 levels result in cristae malformation including reduced respirasome levels and the ETC complexes activity.