Fig 1: Mtln-KO Mice Exhibit Alterations in Mitochondrial Metabolism, Ca2+ Retention, and Protein Complex Assemblies(A) Permeabilized muscle fibers (cardiac, left ventricular [LV], and skeletal muscle [SKM], gastrocnemius) were harvested from fed or fasted (24 hr) WT or Mtln-KO mice, and oxygen consumption rates (OCR) were measured during sequential addition of palmitoyl-carnitine/malate (PC + Mal), 1 mM ADP (Max ADP), and oligomycin; data are plotted as mean ± SEM. Fed: WT n = 7 (4 males, 3 females), KO n = 7 (3 males, 4 females); fasted: n = 4 females for both WT and KO.(B) Mitochondrial Ca2+ retention capacities were measured in permeabilized LV fibers from fasted female mice (n = 4/genotype) and plotted as mean ± SEM; **p = 0.01.(C) In-gel CI activity assay was performed on cardiac tissue mitochondrial lysates from fed WT female mice (n = 3–4/genotype, representative gels shown) (left). Red and green arrows denote bands with decreased or increased (respectively) CI activity in KO mice. Top red arrow points to a doublet band in WT hearts, with the top band virtually absent in KO mice. BN-PAGE and western blot for CI subunit NDUFA9 was performed on the same samples (right). See Figure S7B for SKM tissue data.(D) Representative BN-PAGE western blot on cardiac tissue mitochondrial lysates shows co-migration of Mtln with various prominent mitochondrial complexes; see Figure S7D for SKM data. Some non-specific bands (e.g., in the KO lane) arise from the secondary antibody.(E) SDS-PAGE (top, cardiac tissue lysates) and BN-PAGE (bottom, cardiac mitochondrial lysates) western blots for proteins involved in the TCA cycle (OGDH), CI assembly (ACAD9), and FAO (ACAD9 and VLCAD). Arrow indicates ACAD9 dimers. n = 3–4; representative blots are shown.(F) IDPR prediction was run on the Mtln protein sequence (56 amino acids) using the three indicated independent algorithms and output scores plotted, along with the mean score (red). Scores above 0.5 indicate predicted IDPRs (orange).(G) SDS-PAGE western blot for Mtln-containing high-molecular-weight (HMW) assemblies in WT and Mtln-KO mouse tissue panels. Nonspecific band provides loading control for comparing WT and KO lanes.