Deletion of heart-type cytochrome c oxidase subunit 7a1 impairs skeletal muscle angiogenesis and oxidative phosphorylation

Abstract

Oxidative metabolism is needed for sustained skeletal muscle function. A key component of such metabolism is cytochrome c oxidase, the 13-subunit terminal complex of the mitochondrial electron transport chain. We used mice null for one of the two isoforms of Cox subunit 7a, heart/skeletal muscle-specific Cox7a1, to examine the cellular and functional responses of muscle adaptation in response to mitochondrial dysfunction. Specifically we determined if deletion of Cox7a1 would (1) limit exercise capacity, and (2) alter genes responsible for skeletal muscle capillarity and mitochondrial biogenesis. Sixteen male mice (Cox7a1 null mice, n = 8, and littermate controls, n = 8) performed incremental and run-to-exhaustion treadmill tests. The hindlimb muscles for both groups were analysed. The results indicated that capillary indices were reduced (by 30.7–44.9%) in the Cox7a1 null mice relative to controls. In addition, resting ATP levels and Cox specific activity were significantly reduced (>60%) in both glycolytic and oxidative muscle fibre types despite an increase in a major regulator of mitochondrial biogenesis, PGC-1β. These changes in the skeletal muscle resulted in exercise intolerance for the Cox7a1 null mice. Thus, our data indicate that deletion of the Cox7a1 isoform results in reduced muscle bioenergetics and hindlimb capillarity, helping to explain the observed impairment of muscle structure and function.

Figure 1. Capillarity is lower in the quadriceps femoris muscle of Cox7a1 null mice, with an increase in fast-myosin heavy chain composition protein expression

Histological representation of capillary profile in Cox7a1 and littermate controls at 20× magnification of the quadriceps muscle (top panels; scale bar, 100 μm). Comparison of N_CAF (the number of capillaries around a fibre, A), C/F_i (capillary-to-fibre ratio on an individual-fibre basis, B), CD (capillary density, C) and CFPE (capillary-to-fibre perimeter exchange, D) between the two groups (n = 3 per group). Representative Western blot results for fast- or slow-myosin heavy chain composition in the quadriceps muscle (E; n = 7 per group). Values are mean ± SEM; *P < 0.001.

Figure 2

The effects of heart/muscle-specific subunit 7a1 deletion on protein expression of angiogenic and anti-angiogenic factors Representative Western blot results from the quadriceps femoris muscle are shown. Western blotting was carried out as described in Methods, using 40 μg muscle homogenate protein loaded onto 7.5% (TSP-1) or 12% (VEGF-A, VEGF-R2, CD47) precast polyacrylamide gels, run for 1 h at 160 V, and electrotransferred onto PVDF membranes. After antibody detection, images of blots were acquired with the Odyssey infrared imaging system and quantitated with its software program (Li-Cor Biosciences). *P < 0.05 (mean ± SEM; 5–6 per group).

Figure 3. Protein expression of mitochondrial regulators PGC-1 proteins and RIP140

Representative Western blot results from the quadriceps femoris muscle were generated as in Fig. 2. PGC-1 (A) was separated on 7.5% gels and RIP140 (B) on 12%. *P < 0.01 (mean ± SEM; 5–8 per group).

Figure 4. Basal ATP levels in both glycolytic and oxidative muscles are reduced in Cox7a1 null mice

Resting ATP concentrations in the quadriceps femoris (A) and soleus (B) muscles were determined from frozen tissue as described in Methods using the boiling method and an ATP bioluminescence kit. *P < 0.001 (mean ± SEM; 8 per group).

Figure 5. Cytochrome c oxidase activity is decreased in quadriceps and soleus muscle of mice lacking heart/muscle-specific subunit 7a1

Cox specific activity was determined in Cox7a1 null mice and littermate controls using the polarographic method by increasing the amount of substrate cytochrome c. Quadriceps muscle, which is more glycolytic (A), and the more oxidative soleus muscle (B), were analysed. Cox specific activity is defined as consumed O₂ (μmol min^-1) standardized to GAPDH levels. Cox activity is presented in arbitrary units (AU, set to 100% at maximal turnover for littermate controls) (mean ± SEM, n = 8 each, *P < 0.001). Note, KCN-insensitive respiration both in the wild-type and the knockout were similar (12.1 and 10.7%, respectively) which is in agreement with values from the literature.