ACUTE REGULATION OF MITOCHONDRIAL ELECTRON TRANSPORT CHAIN PROTEINS: REVEALING THE RELATIONSHIP BETWEEN DYNAMIC PROTEIN ORGANIZATION, PERMEABILITY TRANSITION AND CHRONIC ISCHEMIA-REPERFUSION INJURY

Abstract

Skeletal muscle and heart have the essential task of contracting to allow for movement and blood pumping for organ perfusion. Mitochondria provide the ATP necessary for cardiac and skeletal muscle contraction by combining ADP and Pi at ATP synthase, or Complex V (CV), using the energy of the electrochemical H+ gradient generated during the movement of electrons down the electron transport chain (ETC). Several studies have shown that ETC protein complexes can interact forming structures called supercomplexes, which have been considered strong candidates in promoting metabolic advantage through the optimization of ATP production. Genetically modified murine models and long-term interventions, such as exercise and restrictive diets, have been connected to the modulation of supercomplex formation. However, the ability of mitochondrial proteins to acutely respond to intracellular signals, dynamically altering ETC protein organization, remains poorly understood. Increases in intracellular Ca2+ result in its mitochondrial uptake, with physiological levels activating mitochondrial ATP production and supraphysiological levels leading to a precipitous loss of function due to the opening of the mitochondrial permeability transition pore (MPTP). Although the deleterious effects of MPTP opening have been well described and implicated in the mechanisms of ischemia-reperfusion injury, the exact molecular identity and structural-temporal mechanism of pore formation remain elusive. We found that acute Ca2+ overload rapidly, in seconds, increased CV dimer (CV2) formation, in concert with mitochondrial swelling, loss of membrane potential, and reduced oxygen consumption rates in isolated mitochondria from murine heart and skeletal muscle. These findings suggest that CV2 plays a critical role in MPTP formation. In addition, inhibition of Cyclophilin D (which modulates MPTP opening) with cyclosporin A prevented CV2 formation and mitochondrial swelling. However, blocking CV2 formation with the Fo ATP synthase inhibitor oligomycin did not prevent mitochondrial swelling, suggesting that other proteins, such as ANT (Adenine Nucleotide Translocator), are also involved in MPTP opening. Inhibiting ANT in its matrix-facing conformation with bongkrekic acid reduced CV2 formation and swelling, while locking ANT in its cytosolic-facing conformation with carboxyatractyloside enhanced CV2 formation and mitochondrial rupture. These findings indicate that both CV2 and ANT are essential for MPTP formation, and their combined activity appears critical for permeability transition. Moreover, we were able to uncover that mitochondria can rapidly change their protein organization in response to acute intracellular signals, with these modifications occurring within seconds. This rapid adaptation highlights the dynamic nature of mitochondrial protein assembly in response to intracellular signals, enables investigations to determine if altered responsiveness of protein reorganization to these signals impairs function, and may have significant implications for therapeutic strategies to improve mitochondrial function and prevent MPTP-related ischemia-reperfusion injury. Innate mitochondrial protein organization and mitochondrial function were investigated in Sickle Cell Disease (SCD), a condition associated with chronic ischemia-reperfusion injury. Our results showed that, while SCD mice had similar skeletal muscle mitochondrial oxygen consumption rates, reactive oxygen species (ROS) production rates, and citrate synthase activity as controls, Complex IV (CIV) expression and supercomplex formation tended lower in SCD. SCD mitochondria were also more sensitive to calcium and prone to swelling, an effect that was alleviated by cyclosporin A, an MPTP inhibitor. These findings suggest that alterations in electron transport chain proteins may precede overt mitochondrial dysfunction in SCD skeletal muscle, with MPTP potentially playing a role in SCD pathology. Overall, my PhD research underscores the importance of fine-tuning acute mitochondrial protein organization in cellular homeostasis, bringing to light previously unrecognized mechanisms of pathology. Moreover, these investigations highlight new opportunities for drug development and non-invasive biomarkers that could improve the management of SCD.