Association between ROS production, swelling and the respirasome integrity in cardiac mitochondria

Although mitochondrial Ca²⁺ overload and ROS production play a critical role in mitochondria-mediated cell death, a cause-effect relationship between them remains elusive. This study elucidated the crosstalk between mitochondrial swelling, ROS production, and electron transfer chain (ETC) supercomplexes in rat heart mitochondria in response to Ca²⁺ and tert-butyl hydroperoxide (TBH), a lipid-soluble organic peroxide. Results showed that ROS production induced by TBH was significantly increased in the presence of Ca²⁺ in a dose-dependent manner. TBH markedly inhibited the state 3 respiration rate with no effect on the mitochondrial swelling. Ca²⁺ exerted a slight effect on mitochondrial respiration that was greatly aggravated by TBH. Analysis of supercomplexes revealed a minor difference in the presence of TBH and/or Ca²⁺. However, incubation of mitochondria in the presence of high Ca²⁺ (1 mM) or inhibitors of ETC complexes (rotenone and antimycin A) induced disintegration of the main supercomplex, respirasome. Thus, PTP-dependent swelling of mitochondria solely depends on Ca²⁺ but not ROS. TBH has no effect on the respirasome while Ca²⁺ induces disintegration of the supercomplex only at a high concentration. Intactness of individual ETC complexes I and III is important for maintenance of the structural integrity of the respirasome.