Age modifies respiratory complex I and protein homeostasis in a muscle type-specific manner

Abstract

Changes in mitochondrial function with age vary between different muscle types, and mechanisms underlying this variation remain poorly defined. We examined whether the rate of mitochondrial protein turnover contributes to this variation. Using heavy label proteomics, we measured mitochondrial protein turnover and abundance in slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) from young and aged mice. We found that mitochondrial proteins were longer lived in EDL than SOL at both ages. Proteomic analyses revealed that age-induced changes in protein abundance differed between EDL and SOL with the largest change being increased mitochondrial respiratory protein content in EDL. To determine how altered mitochondrial proteomics affect function, we measured respiratory capacity in permeabilized SOL and EDL. The increased mitochondrial protein content in aged EDL resulted in reduced complex I respiratory efficiency in addition to increased complex I-derived H2 O2 production. In contrast, SOL maintained mitochondrial quality, but demonstrated reduced respiratory capacity with age. Thus, the decline in mitochondrial quality with age in EDL was associated with slower protein turnover throughout life that may contribute to the greater decline in mitochondrial dysfunction in this muscle. Furthermore, mitochondrial-targeted catalase protected respiratory function with age suggesting a causal role of oxidative stress. Our data clearly indicate divergent effects of age between different skeletal muscles on mitochondrial protein homeostasis and function with the greatest differences related to complex I. These results show the importance of tissue-specific changes in the interaction between dysregulation of respiratory protein expression, oxidative stress, and mitochondrial function with age.

Keywords: aging; mitochondria; mitochondrial dysfunction; protein turnover; proteome; skeletal muscle.

Figure 1

Mitochondrial Protein Half‐life is Modified by Age and is Dependent on Muscle Type. (A) Plots showing significant changes in mitochondrial protein half‐life with age. Changes were roughly half increasing and half decreasing in half‐life in EDL, while the majority of mitochondrial proteins in SOL increased half‐life (slower turnover rate). Proteins with half‐life longer than one year were excluded from the analyses. (B) Venn diagrams showing few mitochondrial proteins decreased half‐life in aged SOL, whereas those that increased half‐life were similar to those that increased half‐life with age in EDL. q value < 0.05.

Figure 2

Protein Abundance is Modified by Age and Muscle Type (A) 137 total proteins significantly (adjusted P value [q value] < 0.05) increased in content with age in EDL among 745 total proteins detected. 108 of these proteins were mitochondrial, comprising over 96% of mitochondrial proteins that changed with age in the EDL. Black ‐ EDL only, dark grey ‐ EDL and SOL, light grey ‐ SOL only. (B) Comparing citrate synthase activity between mitochondrial enriched fractions and whole cell homogenate shows that the efficiency of extraction was not different in young vs. aged tissues and was not different between EDL and SOL in either age group. (C) Measurement of the AUC of all mitochondrial proteins identified using on an online database (mitoP2, http://www.hsls.pitt.edu/obrc/index.php?page=;URL1097158105) relative to total AUC indicates that mitochondrial content of aged EDL is increased relative to young EDL. * P < 0.05. (D) The direction of change with age was often different between EDL and SOL. Heat map of old/young ratio of protein abundance grouped by Ingenuity Pathway Analyses (IPA) canonical pathways. Top three pathways shown, ordered left to right by the significance of the pathway change with age, q < 0.05 increased with age in darker red, and decreased with age in darker blue. Some proteins occur in more than one pathway. EDL top, SOL bottom rows. IPA of Proteins that change abundance with age, q < 0.05, excluding pathways with less than 4 gene products that changed with age are listed in Table S2.

Figure 3

Age Causes Changes in Respiratory Capacity and Mitochondrial Content. Respiration in fast twitch EDL (A) was maintained with age, but decreased in slow twitch SOL (B), n = 12─24. Mitochondrial respiratory protein content of representative subunits increased with age in EDL (C) while stable or decreased in SOL (D), n = 10─17. Inset shows western analyses of respiratory components CI subunit NDUFB8, CII 30 kDa subunit, and CIV subunit I. 42 kD Ponceau (actin) band was used to normalize protein load. Data are expressed relative to young EDL or SOL. Respiratory flux per mitochondrial content is expressed as a fraction of the young control value (E and F), n = 8. * P < 0.05, *** P < 0.001.

Figure 4

Effect of Age on Expression and Half‐life of ETS Components in the EDL. Map of complex I respiratory apparatus, electron transfer flavin (ETF) moiety and table of other respiratory complexes (mitochondria‐encoded in bold) showing changes in protein abundance (Ab) and half‐life (t_1/2). *NDUFA4, traditionally associated with complex I, may be associated instead with complex IV (Balsa et al. 2012). n.d. = not detected.

Figure 5

Improved Age Related Dysfunction of Complex I Respiratory Capacity, Efficiency and Protein Content in mCAT Mice. Respiration in aged mCAT mice (A) was elevated in EDL and (B) not significantly changed in SOL compared to WT, n = 5─12. (C) Complex I content of aged EDL in mCAT mice is decreased compared to WT aged mice (aged from Fig. 3C). Inset shows western analyses of CI subunit NDUFB8. 42 kD Ponceau (actin) band was used to normalize protein load. Shown relative to young EDL or SOL, n = 11. (D) Complex I respiratory flux per mitochondrial content of NdufB8 is expressed as a fraction of the young control value (aged from Fig. 3D). All data sets in figure D were significantly different than hypothetical mean of 1 (young) with the exception of aged mCAT EDL, which was not different than young, but was significantly different from aged WT mice (T‐test), n = 8. * P < 0.05, ** P < 0.01 compared to WT.

Figure 6

Schematic Model of Mitochondrial Dysfunction with Age. Mitochondrial protein quality control declines in susceptible tissue while oxidative stress increases with age. This leads to mitochondrial dysfunction presented by accumulation of mitochondrial respiratory proteins to compensate for decreased mitochondrial quality. Mitochondrial‐targeted antioxidant (mCAT) improves protein quality with age resulting in improved mitochondrial function.