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Skeletal muscle mitochondrial interactome remodeling is linked to functional decline in aged female mice

Abstract

Genomic, transcriptomic and proteomic approaches have been used to gain insight into molecular underpinnings of aging in laboratory animals and in humans. However, protein function in biological systems is under complex regulation and includes factors besides abundance levels, such as modifications, localization, conformation and protein–protein interactions. By making use of quantitative chemical cross-linking technologies, we show that changes in the muscle mitochondrial interactome contribute to mitochondrial functional decline in aging in female mice. Specifically, we identify age-related changes in protein cross-links relating to assembly of electron transport system complexes I and IV, activity of glutamate dehydrogenase, and coenzyme-A binding in fatty acid β-oxidation and tricarboxylic acid cycle enzymes. These changes show a remarkable correlation with complex I respiration differences within the same young–old animal pairs. Each observed cross-link can serve as a protein conformational or protein–protein interaction probe in future studies, which will provide further molecular insights into commonly observed age-related phenotypic differences. Therefore, this data set could become a valuable resource for additional in-depth molecular studies that are needed to better understand complex age-related molecular changes.

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Fig. 1: Experimental workflow.
Fig. 2: Quantitative cross-linking enables detection of reproducible changes in the interactome of aging mitochondria.
Fig. 3: Assembly of CI and CIV integrity is affected in aging muscle.
Fig. 4: Cross-link levels associated with DHE3 activation are decreased in aged mitochondria.
Fig. 5: TCA cycle and FAO.
Fig. 6: Interactome remodeling associated with changes in muscle metabolism with aging.

Data availability

Mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE repository under identifiers PXD031643 and PXD031644.

Cross-linking data have been uploaded to the XLinkdb online database (http://xlinkdb.gs.washington.edu/xlinkdb/Interactome_of_aged_muscle_mitochondria.php).

Code availability

R markdown used for statistical analysis and figure generation is available at https://github.com/brucelab/aging_mito_interactome_data_analysis.

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Acknowledgements

Figures were created with BioRender.com. We thank members of the Bruce laboratory for helpful discussion. This work was funded by National Institutes of Health grants P01-AG001751 (D.J.M. and M.D.C.), R56-AG070096 (D.J.M. and J.E.B.), T32 AG066574 (G.A.P.), R35GM136255 (J.E.B.) and R01HL144778 (J.E.B.).

Author information

Authors and Affiliations

Authors

Contributions

A.A.B., G.A.P., D.J.M. and J.E.B. designed the experiments. A.A.B., G.A.P., D.J.M. and J.E.B. wrote and edited the manuscript. G.A.P., M.D.C. and R.S.S. performed animal experiments. A.A.B. performed cross-linking experiments, mass spectrometry raw data acquisition and processing. A.K. developed computational tools to support structural protein analysis and cross-linking quantitation. D.J.M. and J.E.B. supervised the project.

Corresponding authors

Correspondence to David J. Marcinek or James E. Bruce.

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The authors declare no competing interests.

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Nature Aging thanks Luca Scorrano, Nicolas Demaurex and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Global quantitative cross-linking measurements.

a. Histogram of calculated Euclidean distances for all intraprotein cross-links mapped to AlphaFold predicted structures. b. Boxplots of number of ions used for each log2 ratio in P1, P2, P3, and P4. X-axis is capped at 40 for readability but there are ratios in each sample with more than 40 ions. c. Histogram of differences between a mean log2 ratios for cross-linked residue pairs based on multiple cross-linked peptides (cross-links that connect the same lysines, but can be identified in differently cleaved or modified peptides) and each cross-linked peptide pair d. Submitochondrial localization of significantly changed interlinks (left) and intralinks (right). e. STRING network of proteins with significantly changed cross-links. f. Isolated gastrocnemius mitochondria oxphos capacity normalized by amount of protein from the 4 young (6 months) and 4 old (30 months) female mice. Mean ± SD.

Source data

Extended Data Fig. 2 Complex I and Complex IV cross-linking analysis.

a. Heatmap of log2 ratios of all NDUS1 and NDUV1 cross-links and interprotein cross-links to other CI subunits. b. Boxplots of all intralinks in CI subunits by a biological replicate. c. Boxplots of crosslinks downregulated in aging and non-changing intralinks based on all 4 biological replicates for CI (NDUV1XLs n = 34; other links n = 112; visualized as median and 25th and 75th percentiles, with whiskers indicating minima and maxima) and CIV (f) Interlinks n = 30; other links n = 14; visualized as median and 25th and 75th percentiles, with whiskers indicating minima and maxima). P values are from Welch two-sided t-test. d. Structure of supercomplex (PDB 5GUP) with cross-linked CI and CIII subunits highlighted (top) and specific CI–CIII cross-links mapped to the subunits (bottom); decreased cross-linked are in green. e. CIII cross-links mapped to a bovine structure. Subunits with decreased intralinks highlighted and zoomed in (right). g. Correlation plots of CI and CIV cross-links changing in aging mitochondria and CII OXPHOS with multiple R-squared displayed.

Source data

Extended Data Fig. 3 Complex I and Complex IV in gel activity measurements show decrease in aged muscle mitochondria.

a. In-gel CI activity assay blot with identified bands. b. Quantification of total CI activity (left; statistical significance determined by unpaired two-tailed student’s t-test; p = 0.1726) and CI activity for each identified band (right; statistical significance determined by Ordinary Two-Way ANOVA with Sidak’s post hoc test; p = 0.0011 age main effect). c. In-gel CIV activity assay blot with identified bands. d. Quantification of total CIV activity (left; statistical significance determined by unpaired two-tailed student’s t-test; p = 0.1219) and CIV activity for each identified band (right; statistical significance determined by Ordinary Two-Way ANOVA with Sidak’s post hoc test; p = 0.0045 age main effect; p = 0.0059 for Band 1 Sidak’s post hoc test). Both activity assays were performed using isolated gastrocnemius mitochondria from young (4–6 month, n = 4) and old (27–29 month, n = 5) NIA C57BL/6 J female mice. Mean ± SD. ns, not significant, **p < 0.01 by Sidak’s post hoc test.

Source data

Extended Data Fig. 4 Antenna specific DHE3 cross-links decreased with aging.

a. Decreased and non-changing cross-link levels in glutamate dehydrogenase highlighted on the volcano plot with Bonferroni corrected p value=0.05. b. Heatmap of all DHE3 cross-linked peptide pairs with each individual peptide sequence shown. Cross-linked lysine residues are in red. c. Correlation plots of DHE3 antenna cross-links changing in aged mitochondria and CII OXPHOS. d. Normalized glutamate stimulated respiration across a range of glutamate concentrations (left; statistical significance determined by Two-Way RM ANOVA with Sidak’s post hoc test; p = 0.0058 age main effect) and maximum respiration capacity with glutamate stimulation (right; statistical significance determined by unpaired two-tailed student’s t-test; p = 0.0009) in young (4–6 mo, n = 4) and old (27–29 mo, n = 4) female NIA C57BL/6 J isolated gastrocnemius mitochondria. e. The kinetics of glutamate stimulated respiration are altered with age (left; nonlinear regression determined by [Agonist] vs. normalized response–Variable slope) in young (4–6 mo, n = 4) and old (27–29 mo, n = 4) female NIA C57BL/6 J isolated gastrocnemius mitochondria. The amount of glutamate required to stimulate 50% respiration calculated from the nonlinear regression is increased in old (right; statistical significance determined by unpaired two-tailed student’s t-test; p = 0.0253). Mean ± SD. *p < 0.05, **p < 0.01.

Source data

Extended Data Fig. 5 TCA cycle and FAO enzymes show decrease in cross-links proximal to CoA binding sites.

a. Fumarate hydratase cross-links mapped to a E. Coli structure (PDB 4HGV). Decreased cross-link levels are shown in the zoomed in square. b. Heatmap of log2 ratios of fumarate hydratase cross-links. c. Boxplots for Acadvl cross-links based on all 4 biological replicates. CoA proximal XLs n = 21; other links n = 46; visualized as median and 25th and 75th percentiles, with whiskers indicating minima and maxima; P-values are from Welch’s two-sided t test.

Source data

Extended Data Table 1 Pairwise combinations of old and young mitochondria to create 4 biological replicates for qXL-MS.

Supplementary information

Supplementary Information

Supplementary results with Fig. 1 and discussion on limitations.

Reporting Summary

Supplementary Data 1

Calculations of log_2 changes for each young/old pair in mito yield, respiration and CS activity.

Source data

Source Data Fig. 2

Quantitative cross-linking data and functional measurements.

Source Data Fig. 3

Quantitative cross-linking data and functional measurements of complex I and complex IV.

Source Data Fig. 4

Quantitative cross-linking data and functional measurements of glutamate dehydrogenase.

Source Data Fig. 5

Quantitative cross-linking data for ACADVL, THIL and SUCA/SUCB.

Source Data Fig. 6

Correlation between quantitative cross-linking and functional measurements; XL clustering.

Source Data Extended Data Fig. 1

Global analysis of quantitative cross-linking measurements.

Source Data Extended Data Fig. 2

Quantitative cross-linking data and functional measurements of complex I and complex IV.

Source Data Extended Data Fig. 4a

Uncropped complex I activity gel.

Source Data Extended Data Fig. 4c

Uncropped complex IV activity gel.

Source Data Extended Data Fig. 3

Quantitation and statistical analysis of in gel CI and CIV activity for young and old mice.

Source Data Extended Data Fig. 4

Quantitative cross-linking data and functional measurements of DHE3.

Source Data Extended Data Fig. 5

Quantitative cross-linking data and functional measurements of ACADVL, THIL and SUCA/SUCB.

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Bakhtina, A.A., Pharaoh, G.A., Campbell, M.D. et al. Skeletal muscle mitochondrial interactome remodeling is linked to functional decline in aged female mice. Nat Aging 3, 313–326 (2023). https://doi.org/10.1038/s43587-023-00366-5

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