The HO-1/CO system regulates mitochondrial-capillary density relationships in human skeletal muscle

Abstract

The heme oxygenase-1 (HO-1)/carbon monoxide (CO) system induces mitochondrial biogenesis, but its biological impact in human skeletal muscle is uncertain. The enzyme system generates CO, which stimulates mitochondrial proliferation in normal muscle. Here we examined whether CO breathing can be used to produce a coordinated metabolic and vascular response in human skeletal muscle. In 19 healthy subjects, we performed vastus lateralis muscle biopsies and tested one-legged maximal O2 uptake (V̇o2max) before and after breathing air or CO (200 ppm) for 1 h daily for 5 days. In response to CO, there was robust HO-1 induction along with increased mRNA levels for nuclear-encoded mitochondrial transcription factor A (Tfam), cytochrome c, cytochrome oxidase subunit IV (COX IV), and mitochondrial-encoded COX I and NADH dehydrogenase subunit 1 (NDI). CO breathing did not increase V̇o2max (1.96 ± 0.51 pre-CO, 1.87 ± 0.50 post-CO l/min; P = not significant) but did increase muscle citrate synthase, mitochondrial density (139.0 ± 34.9 pre-CO, 219.0 ± 36.2 post-CO; no. of mitochondrial profiles/field), myoglobin content and glucose transporter (GLUT4) protein level and led to GLUT4 localization to the myocyte membrane, all consistent with expansion of the tissue O2 transport system. These responses were attended by increased cluster of differentiation 31 (CD31)-positive muscle capillaries (1.78 ± 0.16 pre-CO, 2.37 ± 0.59 post-CO; capillaries/muscle fiber), implying the enrichment of microvascular O2 reserve. The findings support that induction of the HO-1/CO system by CO not only improves muscle mitochondrial density, but regulates myoglobin content, GLUT4 localization, and capillarity in accordance with current concepts of skeletal muscle plasticity.

Keywords: GLUT 4; V̇o2max; carbon monoxide; heme oxygenase-1; mitochondrial biogenesis; myoglobin; nuclear respiratory factor-1; oxygen uptake.

Copyright © 2015 the American Physiological Society.

Figures

Fig. 1.

Activation of the heme oxygenase-1 (HO-1)/carbon monoxide (CO) system by CO leads to increases in mtDNA copy number. A: analysis of vastus lateralis (VL) muscle for HO-1 by Western blot pre- (Pre) and postexposure (Post) in typical subjects in groups A–D as shown in Table 1. Densitometry and normalization to β-actin (means ± SE) show an approximately twofold increases in HO-1 protein levels in CO-exposed (groups A and C) compared with air breathing (group B) or altitude exposure (group D) control subjects. *P < 0.05, pre- vs. postexposure. B: mtDNA copy number shows significant postexposure changes only in groups A and C. *P < 0.05 pre- vs. postexposure.

Fig. 2.

Transcriptional regulators of mitochondrial biogenesis pre- and postexposure for 5 days (groups A and C) compared with air control (group B) and altitude exposure control subjects (group D). A: representative Western blots for total VL muscle protein content for NRF-1, GABPA, and PGC-1α in single individuals in each group. B–D: corresponding densitometry histograms for all subjects as means + SE. *P < 0.05, pre- vs. postexposure.

Fig. 3.

Levels of nuclear-encoded mitochondrial proteins for mitochondrial biogenesis in VL muscle samples. A: Polγ, Tfam, and Tfb2m protein levels by Western blot analysis by exposure group. B–D: corresponding means ± SE densitometry values shown in the histograms for all subjects. All 3 proteins were significantly increased in CO-exposed, but not in air breathing or altitude exposure controls. Muscle Polγ, Tfam, and TFB2M protein levels increased as much as twofold after the CO breathing protocols but were unaffected in the muscle of air breathing or altitude exposure control subjects. *P < 0.05, pre- vs. postexposure.

Fig. 4.

Nuclear and mtDNA-encoded mRNA selected for analysis by qRT-PCR of VL muscle. A and B: histograms for relative expression of nuclear-encoded mRNA for cytochrome c (Cyt c) and COX IV from subjects in groups A–D. C and D: histograms of mRNA levels for mtDNA-encoded genes. C shows COX subunit I and D shows ND1. The levels of all 4 transcripts were increased after CO, but not after air breathing or altitude exposure control protocols. Data are means + SE; *P < 0.05 pre- vs. postexposure.

Fig. 5.

Changes in muscle mitochondrial mass by immunofluorescence analysis of citrate synthase (CS). A: CS fluorescence of VL muscle sections for typical subjects in groups A–D (A–D, respectively) pre- and postintervention for each group. Notable increases in the distribution of punctate cytoplasmic staining for CS were observed only after CO and not after the air breathing or altitude exposure control protocols. Note that CO did not affect CS in all fibers; about one-third of the fibers appear unchanged after the exposures. Graphical data (histogram) represent arbitrary fluorescence intensity units for CS staining presented as means + SE. *P < 0.05 pre- vs. postexposure. B: myosin-7 isoform staining of oxidative fiber types in representative sections from muscle tissue of 1 subject after intervention in each of the 4 groups. Overlay of images shows colocalization of myosin-7 and CS in the fibers. The density of CS was increased by CO in committed fibers.

Fig. 6.

Representative transmission electron micrographs showing mitochondria in longitudinal muscle fibers in VL muscle from subjects in groups A–D pre- and postintervention. A: photomicrographs labeled for groups A–D. CO increased the number of organelles in densely packed areas of subsarcolemmal mitochondria (SS, arrow) adjacent to a capillary (C) and also the interconnected lines of intermyofibrillar mitochondria (IFM) between myofibrils adjacent to the cell borders. B: analysis of the number of mitochondrial profiles obtained from 5 electron microscopic fields and 3 grids per subject in each group. Data are means + SE. *P < 0.05, pre- vs. postexposure.

Fig. 7.

Mitochondrial structural gene mRNA expression levels. Four mitochondrial network structural genes were selected for analysis by qRT-PCR: FIS1 (A), MFN1 (B), MFN2 (C), and OPA1 (D). The 5-day CO breathing protocol significantly increased the transcript levels of MFN2 and OPA1, but not MFN1 and FIS1. No significant effect was seen in air breathing or altitude exposure control subjects. *P < 0.05 pre vs. postexposure.

Fig. 8.

Enhanced GLUT4 and muscle myoglobin content after CO exposure. A: immunofluorescence microscopy was used to visualize GLUT4 translocation to the plasma membrane for groups A–D pre- and postexposure. B: graphical data represent arbitrary fluorescence units for intensity of GLUT4 staining. *P < 0.05 for post- vs. pre-GLUT4 localization in CO-exposed and altitude groups. C: histograms of means + SE muscle GLUT4 protein by Western blot analysis after normalization to β-actin in all subjects (groups A–D). GLUT4 increased compared with preexposure levels in CO-exposed subjects, but not in air breathing or altitude exposure control subjects. *P < 0.05 post- vs. preexposure. D: Western blot analysis and histograms for muscle myoglobin content. Densitometry and normalization to β-actin shows approximate doubling of myoglobin (Mb) in CO-exposed subjects (groups A and C) compared with controls. Values are means + SE; *P < 0.05 post vs. preexposure.

Fig. 9.

Analysis of capillarity in VL human skeletal muscle. A: fixed muscle sections were immunostained for CD31 (red) and counterstained for CS (green). Merged images for CD31- and CS-stained sections are shown for pre- and poststudy biopsies from 1 subject in each of the 4 groups. B: quantification of CD31-positive capillaries per myofiber in VL muscle. Histogram data are means + SE for n = 5–8 in each group. *P < 0.05; values in groups A and C are significantly different after CO exposure. C: histogram of means + SE muscle VEGF protein by Western blot analysis after normalization to β-actin in all subjects. *P < 0.05 post vs. preexposure.

Fig. 10.

Representative plots of workload vs. O2 uptake (V̇o2). For the subject in A, V̇o2 continued to rise until the subject reached exhaustion. The extraneous point was recorded soon after cessation whereas V̇o2 and heart rate were still elevated but the load had been removed. The subject in B also demonstrated initially matched rises in V̇o2 and workload, but continued pedaling past the point where the rise in V̇o2 slowed and then reached a plateau, a traditional indicator of a maximal V̇o2 V̇o2max.