Mitochondrial content and distribution changes specific to mouse diaphragm after chronic normobaric hypoxia

Abstract

Chronic hypoxia reduces aerobic capacity (mitochondrial content) in limb skeletal muscles, and one of the causes seems to be decreased physical activity. Diaphragm and other respiratory muscles, however, may have a different pattern of adaptation as hypoxia increases the work of breathing. Thus, we hypothesized that chronic hypoxia would not reduce mitochondrial content in mouse diaphragm. Adult male C57BL/6J mice were kept in normoxia (Fi(O(2)) = 21%, control) or normobaric hypoxia (Fi(O(2)) = 10%, hypoxia) for 1, 2, and 4 wk. Mice were then killed, and the diaphragm and gastrocnemius muscles collected for analysis. In the diaphragm, cytochrome c oxidase histochemistry showed less intense staining in the hypoxia group. The total content of subunits from the electron transport chain, pyruvate dehydrogenase kinase 1 (PDK1), and voltage-dependent anion channel 1 (VDAC1) was evaluated by Western blot. These proteins decreased by 25-30% after 4 wk of hypoxia (P < 0.05 vs. control for all comparisons), matching a comparable decrease in diaphragmatic mitochondrial volume density (control 33.6 +/- 5.5% vs. hypoxia 26.8 +/- 6.7%, P = 0.013). Mitochondrial volume density or protein content did not change in gastrocnemius after hypoxia. Hypoxia decreased the content of peroxisome proliferator-activated receptor gamma (PPARgamma) and PPARgamma cofactor 1-alpha (PGC-1alpha) in diaphragm but not in gastrocnemius. PGC-1alpha mRNA levels in diaphragm were also reduced with hypoxia. BCL2/adenovirus E1B interacting protein 3 (BNIP-3) mRNA levels were upregulated after 1 and 2 wk of hypoxia in diaphragm and gastrocnemius, respectively; BNIP-3 protein content increased only in the diaphragm after 4 wk of hypoxia. Contrary to our hypothesis, these results show that chronic hypoxia decreases mitochondrial content in mouse diaphragm, despite the increase in workload. A combination of reduced mitochondrial biogenesis and increased mitophagy seems to be responsible for the decrease in mitochondrial content in the mouse diaphragm after hypoxia.

Fig. 1.

Cytochrome c oxidase histochemistry shows less intense staining in hypoxic diaphragm [hypoxia, 4 wk (H-4W) and hypoxia, 2 wk (H-2W)] compared with the normoxic controls (n = 4 in each group). No differences were observed after either 2 wk of hypoxia in diaphragm or in gastrocnemius (scale bars = 50 μm).

Fig. 2.

Representative Western blots and densitometries of respiratory complexes (A) and selected mitochondrial proteins (B) in diaphragm and gastrocnemius muscles collected from mice after normoxia (control) or H-2W or H-4W. The content of all of the proteins was significantly reduced after 4 wk of hypoxia (P < 0.05, n = 8 in each group). Values are given as means ± SE.

Fig. 3.

A: mitochondria volume density decreased in the diaphragm but not in gastrocnemius after 4 wk of hypoxia. Representative electron micrographs of diaphragm (top: scale bar = 1 μm) and gastrocnemius (bottom: scale bar = 2 μm). Some of the mitochondria are labeled with white arrows. B: there is a noticeable reduction in mitochondrial density after 4 wk of hypoxia in diaphragm (*P = 0.013, n = 4 in each group), but no change in the gastrocnemius muscle. Values are given as means ± SE.

Fig. 4.

Electron micrographs showing abundant subsarcolemmal mitochondria in diaphragm muscle fibers after hypoxia (arrows, scale bar = 2 μm). Top: typical distribution of mitochondria and capillaries (c) in control mice. After 4 wk of hypoxia, large clusters of mitochondria (arrows) are observed under the sarcolemma that projects and encircles adjacent capillaries (bottom).

Fig. 5.

A: Quantitative RT-PCR of mitochondrial biogenic factors. There was a decreased expression of PGC-1α in diaphragm after 1, 2, and 4 wk of hypoxia (H) compared with the control normoxia levels. No change was observed in gastrocnemius. There was no difference in any of the other biogenic factors evaluated (i.e., TFAM, NRF1, or NRF2) in diaphragm or gastrocnemius (n = 6 in each group). Western blot analysis of PGC-1α (B) and PPARγ (C) revealed decreased protein contents in diaphragm after 2 and 4 wk of hypoxia (H-2W and H-4W respectively) compared with control (n = 6 in each group). Values are expressed as means ± SE; *P < 0.05 for indicated comparisons.

Fig. 6.

BNIP-3 expression was higher after 1 wk of hypoxia (H-1W) in diaphragm (A) and after 2 wk of hypoxia (H-2W) in gastrocnemius (B). LC-3, ATG-5, and ATG-7 mRNA levels were unchanged in either diaphragm or gastrocnemius. B: BNIP-3 protein content in diaphragm was increased after 4 wk of hypoxia of hypoxia (H-4W) compared with control (C) and 2 wk-hypoxia (H-2W) groups (*P < 0.05). No change in BNIP-3 protein content was observed in gastrocnemius. C: Electron micrographs of diaphragm fibers from mice after 4 wk of hypoxia. There were vesicles consistent with autophagosomes (top: arrows). Higher magnification views of these vesicles are shown in the lower panels (scale bar = 0.2 μm for all the images). Values are given as means ± SE (n = 6 in each group).