Mitochondrial Membrane, Outer

Quadruple immunofluorescence was carried out on transverse muscle sections (10 μm) using antibodies detecting subunits of OXPHOS complexes (Supplementary Table S1). Complex I was detected using an antibody against subunit NDUFB823 (link), and Complex IV using an antibody to mtDNA encoded subunit I (COX-I). Mitochondrial mass was quantified using an antibody to porin, an outer mitochondrial membrane voltage-gated ion channel. Laminin, a basement membrane glycoprotein, was used to label the myofibre boundaries (Supplementary Table S1). Briefly, the sections were fixed in cold 4% paraformaldehyde (Sigma) for 3 min and permeabilised in a methanol (Fisher) gradient (10 min 70% methanol, 10 min 95% methanol, 20 min 100% methanol, 10 min 95%.methanol and 10 min 70% methanol). Non-specific protein interactions were blocked with 10% normal goat serum (Sigma) and incubated with the primary antibodies in a humidified chamber at 4 °C overnight (Supplementary Table S1). Following washes in TBST (Sigma), the sections were incubated with the secondary antibodies for 2 h at 4 °C and subsequently with streptavidin conjugated with Alexa 647 (Life Technologies) for 2 h at 4 °C (Supplementary Table S1). The sections were washed and mounted in Prolong Gold (Sigma). No-primary antibody controls, incubated only with anti-laminin antibody, were processed for each muscle sample.

For details on fixation and labelling of isolated cardiomyocytes and tissue sections, refer to the Supplementary Data file. Confocal and STED images of RV sections dual-labelled with translocator of the outer mitochondrial membrane/ryanodine receptors (TOM20/RyR2) were obtained with an Olympus IX83 Abberior Facility Line STED microscope using a 60× oil immersion objective lens (NA 1.42). To show a larger portion of the tissue being analysed, confocal images were first captured with a 70 µm × 70 µm frame size at 80 nm pixel resolution. Then, both confocal and STED images were captured from a smaller portion of the tissue section (15 µm × 15 µm frame size) at a 15 nm pixel resolution with 594 nm and 640 nm lasers simultaneously at excitation laser powers 3–6% for confocal and STED images. Power for the STED depletion laser, emitted at 775 nm, was between 6% and 10%. Furthermore, confocal images of RV sections co-labelled for F-actin (Alexa Fluor 488 Phalloidin conjugate, 1:50, A12379, Thermofisher Scientific, Waltham, MA, USA) and mitochondria (TOM20) were obtained with a Zeiss LSM800 laser-scanning confocal microscope using a 63× oil-immersion objective lens (NA of 1.4). Images were captured at a 50 nm pixel resolution with 488 nm and 594 nm lasers simultaneously at 0.4% laser power.

SLC25A20 structural models have been inserted in a bilayer phospholipid membrane mimicking the inner mitochondrial membrane (IMM), using the web server CHARMM-GUI (http://www.charmm-gui.org, accessed on 1 December 2022) [62 (link)]. The membrane composition was based on the IMM model published by the CHARMM-GUI team available at the CHARMM-GUI Archive (https://charmm-gui.org/?doc=archive&lib=biomembrane, accessed on 1 December 2022) [63 (link)]. Different concentrations and lipid tail composition are used to better represent the inner and outer IMM leaflets. In this model membrane, phosphatidylcholine is the most represented phospholipid species, followed by phosphatidylethanolamine and cardiolipin, the latter being more abundant in the inner layer. Water molecules, from the TIP3P model, were added on both sides of the membrane, forming two layers each 22.5 Å thick. The total system charge was neutralized by adding NaCl ions, reaching a physiological concentration of 0.15 M. The CHARMM36m force field [64 (link)] and the AMBER22 package [65 ] were used to perform the MD simulations of the assembled systems (~100,000 atoms, see Supplementary Table S6 for representative compositions), following the CHARMM-GUI protocol. First, an energy minimization procedure, involving 2500 steps of steepest descent and 2500 steps of conjugate gradient, was performed. Positional restraints were applied on the protein residues (10 kcal mol⁻¹ Å⁻²) and on the membrane (2.5 kcal mol⁻¹ Å⁻²). The resulting minimized systems have then been simulated using the canonical NVT ensemble, reaching a final temperature of 310.15 K. Thereafter, isothermal-isobaric NPT ensemble simulations have been performed to equilibrate the pressure to 1 bar. During the thermalization and equilibration phases, the positional restraints have been gradually reduced. The equilibrated system, without restraints, was properly simulated using the NPT ensemble for a total of 1 μs. Each system was simulated in replica. The Langevin thermostat has been used for both NVT and NPT ensembles, while the Monte Carlo barostat with a semiisotropic pressure scaling has been used for pressure control [66 (link)]. All of the MD simulations have been performed in a periodic boundary system. For the long range non-bonded interactions, the Particle Mesh Ewald method [67 (link)] and a 12 Å cut-off, with a force switching region at 10 Å, were used. The MD simulations were performed with a time step of 2 fs, apart from the apoSLC25A20 simulation for which the hydrogen mass repartitioning (HMR) method [68 (link)] was used with a 4 fs time step.

Muscle samples collected into ice-cold BIOPS and stored on ice or at 4 °C were analyzed for mitochondrial oxidative phosphorylation (P) and electron transfer system capacities (E) using high-resolution respirometry within 24 h of collection. Immediately prior to analysis, samples were saponin permeabilized as described previously [5 (link)]. Permeabilized fibers were then rinsed in mitochondrial respiration solution (MiR05; 110 mM sucrose, 60 mM potassium lactobionate, 0.5 mM EGTA, 3 mM MgCl₂∙6H₂O, 20 mM taurine, 10 mM KH₂PO₄, 20 mM HEPES, 1 g/L BSA, pH 7.1) for 10 min at 4 °C. Approximately 1.5–2.5 mg (wet weight) of rinsed fibers were then immediately added to each chamber of an Oroboros Oxygraph-2k (O2k; Oroboros, Innsbruck, Austria) containing MiR06 (MiR05 + 280 U/mL catalase) and 20 mM creatine. Chambers were maintained at 37 °C and in hyperoxic conditions (200 to 650 µM O₂) through the addition of 200 mM H₂O₂. The previously described [13 (link)] substrate uncoupler inhibitor titration protocol for this study was as follows: (1) complex I substrates, pyruvate (5 mM), and malate (1 mM), to determine mitochondrial proton leak (LEAK); (2) adenosine diphosphate (ADP; 2.5 mM), to quantify complex I-supported P (P_CI); (3) glutamate (10 mM), an additional complex I substrate (P_CIG); (4) cytochrome c (cyt c, 10 µM), to measure integrity of the outer mitochondrial membrane; (5) the complex II substrate, succinate (10 mM), to measure maximal P (P_CI+II); (6) uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 0.5 µM steps), to attain maximal noncoupled E (E_CI+II); (7) a complex I inhibitor, rotenone (0.5 µM), to measure complex II-supported E (E_CII); and (8) a complex III inhibitor, antimycin A (2.5 µM), to quantify non-mitochondrial residual O₂ consumption. All data were normalized to residual O₂ consumption. Respiration data are presented either relative to tissue weight (integrative), CS activity (mitochondrial volume density; intrinsic), or as a ratio of contribution to maximal electron transfer capacity (flux control ratio, FCR).