CISD3/MiNT is required for complex I function, mitochondrial integrity, and skeletal muscle maintenance

Abstract

Mitochondria play a central role in muscle metabolism and function. A unique family of iron-sulfur proteins, termed CDGSH Iron Sulfur Domain-containing (CISD/NEET) proteins, support mitochondrial function in skeletal muscles. The abundance of these proteins declines during aging leading to muscle degeneration. Although the function of the outer mitochondrial CISD/NEET proteins, CISD1/mitoNEET and CISD2/NAF-1, has been defined in skeletal muscle cells, the role of the inner mitochondrial CISD protein, CISD3/MiNT, is currently unknown. Here, we show that CISD3 deficiency in mice results in muscle atrophy that shares proteomic features with Duchenne muscular dystrophy. We further reveal that CISD3 deficiency impairs the function and structure of skeletal muscles, as well as their mitochondria, and that CISD3 interacts with, and donates its [2Fe-2S] clusters to, complex I respiratory chain subunit NADH Ubiquinone Oxidoreductase Core Subunit V2 (NDUFV2). Using coevolutionary and structural computational tools, we model a CISD3-NDUFV2 complex with proximal coevolving residue interactions conducive of [2Fe-2S] cluster transfer reactions, placing the clusters of the two proteins 10 to 16 Å apart. Taken together, our findings reveal that CISD3/MiNT is important for supporting the biogenesis and function of complex I, essential for muscle maintenance and function. Interventions that target CISD3 could therefore impact different muscle degeneration syndromes, aging, and related conditions.

Keywords: Duchenne muscular dystrophy; NDUFV2; NEET/CISD proteins; complex I; mitochondria.

Fig. 1.

Generation and characterization of Cisd3^−/− mice. (A) Targeting of exon 2 of Cisd3 using a flox strategy. (B) PCR analysis of wild-type (WT), floxed, and the knockout (KO) alleles of Cisd3 in transgenic mice. (C) Representative images of a WT and a Cisd3^−/− mice (Left) and box-and-whisker plots showing the weight of WT and Cisd3^−/− mice at age 44 to 47 wk old (Right; SI Appendix, Fig. S1). (D) Representative protein blots (Left) and box-and-whisker plots showing quantitative analysis (Right) for the levels of CISD1, CISD2, and CISD3 proteins in WT and Cisd3^−/− mice. (E and F) Box-and-whisker plots showing the amount of time wild-type (WT) or Cisd3^−/− mice can hang from a wheel (E) or a rod (F). Results are shown for male and female separately and presented as box-and-whisker plots and include all data points of six different animals (three different males and three different females) from each group (WT and Cisd3^−/− mice). Two-way ANOVA followed by a Tukey test was used to calculate statistical significance. White box and white square, WT male mice; gray box and black square, Cisd3^−/− male mice; white box and white circle, WT female mice; gray box and black circle, Cisd3^−/− female mice. Abbreviations: CISD, CDGSH Iron Sulfur Domain; KO, knock out; WT, wild type.

Fig. 2.

Impaired structure and function of mitochondria from skeletal muscles of Cisd3^−/− mice. (A) Representative transmission electron microscope (TEM) images of mitochondria from wild-type (WT) and Cisd3^−/− mice (Top) and box-and-whisker plots showing quantification of mitochondrial damage (Bottom). (B–E) Bar graphs for basal (B), maximal (C), and spare (D) respiration, and glycolytic activity (E) of skeletal muscle fibers from WT and Cisd3^−/− mice (measured with an XFe24 Seahorse apparatus). Results are shown for male mice and presented as box-and-whisker plots and include all data points of six different animals from each group (WT and Cisd3^−/− mice). Two-way ANOVA followed by a Tukey test was used to calculate statistical significance. White box and white square, WT male mice; Gray box and black square, Cisd3^−/− male mice; white box and white circle, WT female mice; Gray box and black circle, Cisd3^−/− female mice. Abbreviations: CISD, CDGSH Iron Sulfur Domain; ECAR, Extracellular Acidification Rate; KO, knock out; OCR, Oxygen Consumption Rate; TEM, transmission electron microscope; WT, wild type.

Fig. 3.

Proteomics analysis of skeletal muscles from wild-type and Cisd3^−/− mice. (A) A diagram showing the number of proteins altered in Cisd3^−/− mice compared to the wild type (WT) in quadricep muscle tissues of 44-wk-old mice. (B) Subcellular localization and gene ontology (GO) annotation of proteins altered in Cisd3^−/− mice compared to WT. (C) Venn diagrams showing the overlap between proteins altered in Cisd3^−/− mice and proteins altered in mice model systems for Duchenne Muscular Dystrophy (DMD), Huntington Disease (HD; R6/2), and Multiple Sclerosis (MS; EAE). (D) Same as in (C) but for mitochondrial proteins. (E–G) Changes in protein abundance in Cisd3^−/− mice are shown for the tricarboxylic acid cycle (TCA; E), fatty acid oxidation (F), and glycolysis/gluconeogenesis (G) pathways. Results are shown for male mice and presented as mean ± SD of three different animals from each group (WT and Cisd3^−/− mice). Two-way ANOVA followed by a Tukey test was used to calculate statistical significance. Abbreviations: CISD, CDGSH Iron Sulfur Domain; DMD, Duchenne muscular dystrophy; EAE, experimental allergic encephalomyelitis; HD, Huntington disease; GO, gene ontology; GO, gene ontology; KO, knock out; MS, multiple sclerosis; WT, wild type; Cs, citrate synthase; Acly, ATP-citrate synthase; Aco2, Aconitase 2; Idh, Isocitrate dehydrogenase [NADP]; Ogdh, Oxoglutarate Dehydrogenase; Suclg, Succinate--CoA ligase; Pc, pyruvate carboxylase; Sdh, Succinate dehydrogenase [ubiquinone]; Fh, Fumarate hydratase; Mdh, Malate dehydrogenase; A-KG, α-Ketoglutarate; Acaa2, 3-ketoacyl-CoA thiolase; Acsl5, Long-chain-fatty-acid—CoA ligase 5; Acad11, Acyl-CoA dehydrogenase family member 11; Acadl, Long-chain specific acyl-CoA dehydrogenase; Echs1, Enoyl-CoA hydratase; Fbp1, Fructose-1,6-bisphosphatase 1; Pck, Phosphoenolpyruvate carboxykinase; Pfkfb1, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 1; Pdk, Pyruvate dehydrogenase (acetyl-transferring)] kinase; Aldoart1, Fructose-bisphosphate aldolase; Mpc, Mitochondrial pyruvate carrier; Vdac3, Voltage-dependent anion-selective channel protein 3, F2.6BP, Fructose 2,6-bisphosphate; DHAP, Dihydroxyacetone phosphate.

Fig. 4.

Identification of proteins associated with CISD3 in skeletal muscles and cluster transfer between CISD3 and NDUFV2. (A) Changes in protein abundance in Cisd3^−/− mice are shown for complex I, II, and III/IV/V proteins. (B) Identification of proteins that associate with CISD3 in vivo in skeletal muscles following immunoprecipitation assays performed with skeletal muscles from wild-type (WT) and Cisd3^−/− mice. Protein identification was performed by proteomics analysis. (C and D) Protein gel (C) and protein blot with antibody against CISD3 (D) showing in vitro protein–protein interaction between CISD3 and NDUFV2. Arrow indicates the location of the protein–protein complex between CISD3 and NDUFV2. Identification of proteins found in the band indicated by arrows in (D) using proteomics analysis is shown below (D). (E) Cluster transfer from holo-CISD3 to apo-NDUFV2 visualized by changes in spectra of the two proteins following coincubation. Results are shown for male mice. Abbreviations: CISD, CDGSH Iron Sulfur Domain; Ndufv2, NADH:ubiquinone oxidoreductase core subunit V2; IB, Immunoblotting; IBAQ, intensity-based absolute quantification. mt-Nd3, NADH-ubiquinone oxidoreductase chain 3; Ndufa, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit; Ndufb10, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10; Ndufs, NADH:ubiquinone oxidoreductase core subunit; Ndufv, NADH:ubiquinone oxidoreductase core subunit V; Sdh, Succinate dehydrogenase [ubiquinone]; Uqcr, Cytochrome b-c1 complex subunit; Cox, Cytochrome c oxidase subunit 5A; Mtatp, ATP synthetase protein; Co-ip, Coimmunoprecipitation; SN, Supernatant; MW, Molecular Weight; CISD, CDGSH Iron Sulfur Domain; KO, knock out; WT, wild type.

Fig. 5.

Coevolutionary analysis of the CISD3–NDUFV2 complex. (A) A general pipeline for extraction and analysis of coevolutionary data: seed sequences for CISD3 and NDUFV2 are used to generate their respective MSAs through phmmer software. Entries from one MSA are paired to entries in the other MSA via PPM and the pairs are concatenated horizontally. The full MSA of concatenated pairs is used in Direct Coupling Analysis to generate ranked DI contacts. A subset of the top DI contacts is used in future simulations and visualizations. (B) Contact map with residue pairs plotted as grid points. DI pairs of intraprotein contacts (CISD3: purple; NDUFV2: orange) and interprotein contacts (CISD3–NDUFV2: dark red) are represented with monomeric native contacts (gray) and SBM+DCA contacts (blue). (C) Visualization in UCSF Chimera (51) of the Amber minimized SBM+DCA model. From the top 50, the top 10 proximal interprotein DI pairs are connected with pseudobonds (green). The minimal distance between 2Fe-2S clusters is approximately 15.7 Å, shown as a black, dashed line. Abbreviations: CISD, CDGSH Iron Sulfur Domain; NDUFV2, NADH:ubiquinone oxidoreductase core subunit V2; DI, direct information; HMMER, biosequence analysis using profile hidden Markov models; MSA, multiple sequence alignment.

Fig. 6.

A model for the function of CISD3 in mitochondria from skeletal muscles of mice. CISD3 is shown to donate its [2Fe-2S] clusters to NDUFV2 (and perhaps other 2Fe-2S proteins of complexes I and II), thereby supporting the function of the mitochondrial respiratory chain in muscle mitochondria and enabling enhanced metabolic activity. Based on previous studies in cancer cells (22, 23), CISD3 is also proposed to function as a part of a [2Fe-2S] cluster relay that transfer clusters from within the mitochondria to the cytosol. In the absence of CISD3 the function of the mitochondrial respiratory chain is reduced, and mitochondria are more prone to iron overload and structural damage. Abbreviations: CISD, CDGSH Iron Sulfur Domain; NDUFV2, NADH:ubiquinone oxidoreductase core subunit V2; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species.