Phosphate Is Essential For Rearrangements In Mitochondrial Electron Transport Chain Proteins During Calcium Overload

Skeletal muscle has the essential task of contracting to allow for movement, and mitochondria provide the ATP necessary by combining ADP and Pi at ATP synthase (Complex V), using the energy of the electrochemical H+ gradient generated during the movement of electrons down the electron transport chain (ETC). A rise in cytosolic Ca++ results in mitochondrial uptake of Ca++, with physiological levels activating mitochondrial ATP production and supraphysiological levels resulting in a precipitous loss of function. PURPOSE: The goal of this study was to determine if activating and overload Ca++ can differentially induce changes in the organization of ETC protein complexes into supercomplexes (SCs), and specifically, the presence of the CV monomer (CV), and, if Pi is essential for these rearrangements. METHODS: Sprague-Dawley rat skeletal muscle mito were isolated using differential centrifugation. Maximal, ADP-stimulated O2 consumption rates were measured in the presence increasing Ca++ concentrations in the presence of glutamate and malate. Mito were then incubated with either activating Ca++ or overload Ca++ in the presence or absence of Pi, followed by solubilization (digitonin) and loading into a gradient BN-PAGE gel to allow for the assessment of ETC SCs. Expression of mito proteins was analyzed via densitometry and two-tailed t-tests. RESULTS: Maximal Ca++-activated JO was obtained with 3.5 μM Ca++. In the absence or presence of Pi, Complex V monomer abundance was higher with Ca++ activation compared to Ca++ overload (22.8 ± 2.2 vs 15.2 ± 1.4, p = 0.004; 21.9 ± 2.7 vs 9.6 + 2.2, p = 0.003). When Pi was added after Ca++ overload, CV abundance decreased from 15.2 + 1.4 to 9.6 + 2.2, p = 0.005. Conversely, overall SCs abundance was higher during Ca++ overload (47.8 + 3.8) than Ca++ activating conditions (32.1 + 2) p = 0.004, in the presence of Pi. No effect was observed on SCs expression between overload (47.8 + 3.8) and activating (35.8 + 4) Ca++ conditions in the absence of Pi (p = 0.12). CONCLUSIONS: Mito supercomplexes can be acutely regulated and Pi seems to be essential for the mito function during Ca++ activation and overload, indicating that Pi levels regulate muscle mito function. Uncovering the relevance of Pi to mito’s dynamic organization of proteins can illuminate on new strategies to improve skeletal muscle function. American Heart Association Scientific Development Grant (16SDG30770015); University of Maryland Faculty-Student Research Award