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Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in the failing murine heart

Nature Cardiovascular Research volume 1, pages 855–866 (2022)Cite this article

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Abstract

Advancements in cross-linking mass spectrometry bridge the gap between purified systems and native tissue environments, allowing the detection of protein structural interactions in their native state. In this study, we used isobaric quantitative protein interaction reporter (iqPIR) technology to compare the mitochondrial protein interactomes in healthy and failing murine hearts 4 weeks after transverse aortic constriction. The failing heart interactome includes 588 statistically significant cross-linked peptide pairs altered in the disease condition. We observed an increase in the assembly of ketone oxidation oligomers corresponding to an increase in ketone metabolic utilization; remodeling of NDUA4 interaction in Complex IV, likely contributing to impaired mitochondrial respiration; and conformational enrichment of the ADP/ATP carrier ADT1, which is non-functional for ADP/ATP translocation but likely possesses non-selective conductivity. Our application of quantitative cross-linking technology in cardiac tissue provides molecular-level insights into the complex mitochondrial remodeling in heart failure while bringing forth new hypotheses for pathological mechanisms.

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Fig. 1: Quantitation of mitochondrial protein interactome in failing hearts.
Fig. 2: Decreased interaction between NDUA4 and C6XB1 affects CIV activity in TAC.
Fig. 3: Active conformational states of ketone oxidation proteins enriched in TAC.
Fig. 4: Enrichment of an intermediate state of ADP/ATP carrier detected in TAC.

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Data availability

Cross-linking data have been deposited at XlinkDB (http://xlinkdb.gs.washington.edu/xlinkdb/index.php) and are publicly available (network name: Caudal_iqPIR_TACsham_Bruce). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE70 partner repository with the dataset identifiers PXD027757 and PXD035622. The following publicly available files were included: PDB 5Z62, PDB 3DLX, PDB 3OXO, PDB 1OKC, PDB 6GCI, PDB 2LCK and UniProt P48962. Any additional information required to reanalyze the data reported in this paper is available from the lead contacts upon reasonable request. All other data supporting the findings in this study are included in the main article and associated files.

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Acknowledgements

We thank all Tian and Bruce laboratory members for their thoughtful discussions and support. We thank the University of Washington Proteomics Resource for advice and helpful discussions. We thank Y.-W. A. Hsu for assistance with the animal models. We thank J. Ritterhoff and F. Drees for their technical support and guidance. This work was supported, in part, by National Institutes of Health (NIH) grants HL110349, HL129510 and HL142628 (to R.T.); HL144778, GM097112, GM086688 and R35GM136255 (to J.E.B); American Heart Association (AHA) Predoctoral Fellowship 20PRE35120126 (to A.C.); AHA Postdoctoral Fellowship 18POST33990352 (to B.Z.); China Scholarship Council Fellowship 202006320416 (to H.C.); and NIH 2T32DK007247-41 and AHA Career Development Award 930223 (to M.A.W).

Author information

Author notes

  1. Hongye Chen

    Present address: Department of Cardiology, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China

  2. These authors contributed equally: Arianne Caudal, Xiaoting Tang.

  3. These authors jointly supervised this work: Rong Tian, James E. Bruce.

Authors and Affiliations

  1. Department of Biochemistry, University of Washington, Seattle, WA, USA

    Arianne Caudal & Rong Tian

  2. Mitochondria and Metabolism Center, Department of Anesthesiology & Pain Medicine, University of Washington, Seattle, WA, USA

    Arianne Caudal, Outi Villet, Hongye Chen, Bo Zhou, Matthew A. Walker & Rong Tian

  3. Department of Genome Sciences, University of Washington, Seattle, WA, USA

    Xiaoting Tang, Juan D. Chavez, Andrew Keller, Jared P. Mohr, Anna A. Bakhtina & James E. Bruce

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Contributions

A.C., X.T., J.D.C., R.T. and J.E.B. designed the experiments. A.C., X.T., J.D.C., A.K., M.A.W., R.T. and J.E.B. wrote the manuscript. A.C., X.T., R.T. and J.E.B. edited the manuscript. A.C., X.T., J.D.C. and A.K. performed formal analysis. A.C., B.Z. and M.A.W. performed animal experiments. A.C. and J.D.C. performed cross-linking experiments. J.D.C. performed protein preparation. J.D.C., X.T. and A.K. performed mass spectrometry raw data acquisition and processing. A.K. developed computational tools to support structural protein analysis and cross-linking quantitation. J.P.M. and A.A.B. performed cross-linking analysis and structural modeling. O.V. and H.C. performed animal surgeries. R.T. and J.E.B. supervised the project.

Corresponding authors

Correspondence to Rong Tian or James E. Bruce.

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Extended data

Extended Data Fig. 1 (a-b) Left ventricle internal dimension and wall thickness in TAC and Sham groups determined by echocardiography four weeks post-surgery.

(c-d) Lung and liver edema (wet weight/dry weight in mg) measured at tissue harvest. (e) Quantitation of mean cross-link (XL) ratio vs mean dead-end (DE) ratio for each cross-linked peptide pair statistically changed in TAC in at least 4/6 biological pairs (Log2 TAC/Sham). The sum of the Mean DE ratio for Protein A and Protein B is shown to account for cross-links between two different proteins. (f) Interaction network of lysine residues (black nodes) connected by observed cross-links (edges). Edges are colored according to increasing (red) and decreasing (blue) quantitation of statistically significant subset of cross-links (Log2 TAC/Sham) shown by color scale. For (a-d), all data are n=12 animals, AVG+/-SEM, *denotes p<0.05 by unpaired, two-tailed Student’s t-test.

Extended Data Fig. 2 (a) Structural insight into CX6B1 R20 forming salt-bridges at NDUA4-CX6B1 interface.

R20 side chain (green) forms a salt bridge with CX6B1 D16 (green) and NDUA4 D60 (black), which pinpoints an interface necessary for the stability of CIV. Side-chains of R20, D16, and NDUA4 D60 (partially resolved) are depicted in stick representation. Cross-linked lysine sidechains are shown as yellow (CX6B1) or orange (NDUA4) spheres. (b) Cytochrome C oxidase enzymatic activity assay in tissue homogenates from TAC and Sham hearts, normalized to Citrate Synthase activity. N=4 animals, AVG+/-SEM, *denotes p<0.05 by unpaired, two-tailed Student’s t-test. (c) Table summarizing the mean cross-linking ratio and DE ratio for cross-linked peptide pairs, including values obtained across biological replicates for cross-links in ADT isoforms. (d) Representative Blue Native-PAGE (BN-PAGE) analysis of mitochondria isolated from Sham and TAC groups. Coomassie stain (left) for total protein loading, In-gel CIV activity stain (middle), with CI activity stain overlay (right). Gels were run in duplicates. (e) Representative BN-PAGE immunoblot of NDUA4 containing SCs from digitonin solubilized isolated mitochondria. Blots were run in duplicates. (f) Representative BN-PAGE immunoblot of NDUA4 containing SCs from DDM solubilized isolated mitochondria. Blots were run in duplicates. (g) Cross-linked peptide pairs (yellow lysine side chains) mapped onto the M-state conformation of ADT1 (PDB: 6GCI). Salt bridges between K96-D196 and K199-D292 contribute to the gating mechanism, which closes the M-state to the IMS and would make lysines unavailable for cross-linking. Aspartic acid sidechains are shown in magenta. (h) Cross-linked peptide pairs (yellow lysine side chains) mapped onto the C-state conformation of bovine ADT1 (PDB: 1OKC). K33 and D231 are known to form a salt bridge that stabilizes the C-state and would make K33 unavailable for cross-linking. Aspartic acid sidechains are shown in magenta.

Source data

Extended Data Fig. 3 (a) AlphaFold-predicted BDH1_MOUSE structure.

Crosslinked residues were indicated in yellow spheres, and crosslinks shown in red lines mean they were increased in TAC samples. (b) The pLDDT plot of the predicted BDH_mouse structure. (c) Alignment of apo (grey, PDB: 3OXO chain A) and substrate-bound (yellow, PDB: 3OXO chain E) monomers of porcine SCOT1. CoA is colored in magenta and bound to the active site. Lysine sidechains are shown in stick representation. Alignment depicts the structural differences between the dynamic C-terminal domain and the static N-terminal domain during substrate-binding. A close-up view specifies cross-linked lysines (K418 and K421). (d) Ketone-driven oxygen consumption rate (OCR) of mitochondria isolated from TAC and Sham hearts. Baseline OCR (State 1) was measured, followed by sequential injections of β-hydroxybutyrate/malate (State 2), ADP (State 3), Oligomycin (State 4μ), and FCCP (FCCPmax). N=6 animals, AVG+/-SEM, *denotes p<0.05 by unpaired, two-tailed Student’s t-test.

Supplementary information

Reporting Summary

Supplementary Video 1

Cross-linking determines ADT1 conformational states

Supplementary Table

Supplementary Data 1: log2 ratios of non-redundant peptide pairs across six biological replicates and their mean log2 ratios showed significant changes between TAC and Sham samples and DE mean log2 ratios of the corresponding proteins. Supplementary Data 2: LFQ of mitochondrial proteins from six biological replicates of TAC and Sham samples and combined log2 ratios of TAC/Sham was reported. Supplementary Data 3: R2 values of pairwise linear regression of six pairs of biological replicates and mean R2 values of FF and FR regression. Supplementary Data 6: log2 ratios of residue pairs across six biological replicates and their mean log2 ratios for each residue pair with P value and 95% confidence level and several distinct peptide pairs reported. Supplementary Data 7: ANOVA test of the quantitation of the same residue pair generated from fully-cleaved peptide pairs and its corresponding missed-cleaved peptide pairs

Supplementary Data 4

SCOT1 octamer structure file generated by superimposition of AlphaFold monomer structures onto 3OXO to provide structural data on missing regions containing K296

Supplementary Data 5

AlphaFold model structure of ADT1 open channel consistent with increased cross-link levels quantified in TAC hearts

Source data

Source Data Extended Data Fig. 2

Uncropped gels for Extended Data Fig. 2c–e

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Caudal, A., Tang, X., Chavez, J.D. et al. Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in the failing murine heart. Nat Cardiovasc Res 1, 855–866 (2022). https://doi.org/10.1038/s44161-022-00127-4

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