Rat diaphragm mitochondria have lower intrinsic respiratory rates than mitochondria in limb muscles

Abstract

The mitochondrial content of skeletal muscles is proportional to activity level, with the assumption that intrinsic mitochondrial function is the same in all muscles. This may not hold true for all muscles. For example, the diaphragm is a constantly active muscle; it is possible that its mitochondria are intrinsically different compared with other muscles. This study tested the hypothesis that mitochondrial respiration rates are greater in the diaphragm compared with triceps surae (TS, a limb muscle). We isolated mitochondria from diaphragm and TS of adult male Sprague Dawley rats. Mitochondrial respiration was measured by polarography. The contents of respiratory complexes, uncoupling proteins 1, 2, and 3 (UCP1, UCP2, and UCP3), and voltage-dependent anion channel 1 (VDAC1) were determined by immunoblotting. Complex IV activity was measured by spectrophotometry. Mitochondrial respiration states 3 (substrate and ADP driven) and 5 (uncoupled) were 27 ± 8% and 24 ± 10%, respectively, lower in diaphragm than in TS (P < 0.05 for both comparisons). However, the contents of respiratory complexes III, IV, and V, UCP1, and VDAC1 were higher in diaphragm mitochondria (23 ± 6, 30 ± 8, 25 ± 8, 36 ± 15, and 18 ± 8% respectively, P ≤ 0.04 for all comparisons). Complex IV activity was 64 ± 16% higher in diaphragm mitochondria (P ≤ 0.01). Mitochondrial UCP2 and UCP3 content and complex I activity were not different between TS and diaphragm. These data indicate that diaphragm mitochondria respire at lower rates, despite a higher content of respiratory complexes. The results invalidate our initial hypothesis and indicate that mitochondrial content is not the only determinant of aerobic capacity in the diaphragm. We propose that UCP1 and VDAC1 play a role in regulating diaphragm aerobic capacity.

Fig. 1.

Respiration rates are lower in diaphragm mitochondria. O₂ consumption rates were normalized to mitochondrial protein. A: states 3 and 5 were significantly lower in diaphragm mitochondria compared with triceps surae (TS). B: respiratory control ratio (RCR) is significantly lower in diaphragm mitochondria (DIA) compared with TS. *P < 0.05 for all comparisons, n = 15 rats.

Fig. 2.

Content of respiratory complexes is higher in diaphragm mitochondria. Representative Western blots and densitometries of respiratory complexes from TS and diaphragm. The content of respiratory complexes (C) III, IV, and V was significantly higher in diaphragm mitochondria. OD, optical density. *P < 0.02 for all comparisons, n = 4 rats.

Fig. 3.

Complex I and IV activities in TS and diaphragm mitochondria. A: complex I activity was not significantly different in diaphragm and TS mitochondria. P > 0.05, n = 6 rats. B: complex IV activity was 64 ± 16% higher in the diaphragm mitochondria compared with TS. *P < 0.01, n = 8 rats.

Fig. 4.

Uncoupling protein (UCP) 1 content is higher in diaphragm mitochondria. Representative Western blots and densitometries of uncoupling proteins, UCP1, UCP2, and UCP3, in mitochondria from TS and diaphragm. The content of UCP1 was 36 ± 15% higher in diaphragm mitochondria. These results indicate that the contents of UCP2 and UCP3 were not different in these muscles. *P < 0.05, n = 4 rats.

Fig. 5.

Voltage-dependent anion channel 1 (VDAC1) is higher in diaphragm mitochondria. Representative Western blots and densitometries of VDAC1 expression levels on mitochondria from TS and diaphragm. VDAC1 content was 18 ± 8% higher in diaphragm mitochondria. *P < 0.05, n = 4 rats.