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 (V̇o_2max) 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 V̇o_2max (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.

the induction of heme oxygenase-1 (HO-1) during the process of heme degradation produces an important physiological gas, carbon monoxide (CO) (28). Physiologically, the HO-1/CO system participates in cellular defenses against inflammation and against oxidative stress (50) but also acts as a redox driver of mitochondrial biogenesis (37, 49). Moreover, the controlled use of CO breathing or administration of CO-releasing molecules pharmacologically mitigates a wide range of pathological injuries in cells, tissues, and whole animals (25, 33, 44). By implication, if the gas can be given safely and effectively, CO could have clinical utility in organ failure syndromes where mitochondrial damage is important.

An increase in tissue CO content in mice by CO breathing increases mitochondrial DNA (mtDNA) transcription, replication, and mitochondrial proliferation (49). This reflects induction of a genetic program for mitochondrial biogenesis and quality control that shares many of the protective features of the HO-1/CO system, including protection against oxidative stress and inflammation (36). In both processes, adaptive pathways that depend on the expression of cytoprotective and anti-inflammatory genes are also activated (33, 44).

After episodes of mitochondrial damage, induction of the HO-1/CO system leads to expansion of mitochondrial volume density, which is essential for cell maintenance and repair processes that require increased energy utilization (48). In an earlier study of low-level CO exposure in humans, our group found that brief, intermittent CO inhalation [100 parts per million (ppm), 1 h/day for 5 days] increased certain molecular regulators of mitochondrial biogenesis in skeletal muscle but did not increase maximal O₂ uptake (V̇o_2max) (43). Those muscle samples demonstrated increases in levels of HO-1 enzyme and mitochondrial transcription factor A (Tfam), which is required for mtDNA transcription and replication, as well as in mtDNA polymerase (Polγ).

Although the findings suggested that muscle plasticity, which varies profoundly with the type and intensity of the stimulus, requires mitochondrial biogenesis (21), the full implications are far from clear. It is known that heavy resistance exercise causes muscle hypertrophy with an increase in strength, but without major changes in the aerobic metabolic machinery, including mitochondrial mass. In contrast, endurance training increases the aerobic capacity mediated by an increase in mitochondrial content (1, 29), but without muscle hypertrophy or improved strength (22). Exercise also induces glucose transport, for example glucose transporter GLUT4, which makes for more rapid glucose uptake and greater glycogen storage after glycogen-depleting exercise.

Despite the link between mitochondrial density and aerobic capacity, low-level CO exposures that stimulated mitochondrial biogenesis in healthy people were not accompanied by changes in systemic V̇o_2max (43). The most plausible explanations involve incomplete induction of the mitochondrial biogenesis program, differences in responsiveness of the molecular and the physiological regulators, incomplete linkage between mitochondrial biogenesis and microvascular reserve, and/or a delay in the change in V̇o_2max due to absence of training. There also may be variable contributions of mitochondrial capacity and systemic O₂ delivery to exercise limitation during different exercise protocols.

By employing a higher CO concentration (200 ppm) and single-leg exercise in the setting of hypobaric hypoxia, we partially addressed this apparent discontinuity, since systemic O₂ delivery assumes a smaller role in limiting the exercise capacity in single-leg exercise at altitude. It is estimated that the role of systemic O₂ delivery decreases from 70–75% with two legs to only 50% with one leg (8). The importance of the two main peripheral components, mitochondrial capacity and local diffusion, has been proposed to increase symmetrically to ∼25% for each. In other words, the site of the limitation is shifted more toward the muscle when exercising a smaller muscle group in single-leg testing. Altitude (acute hypobaric hypoxia) similarly favors an increase in limitation due to peripheral components (9). Peripheral diffusion in particular has been shown to be highly dependent on red blood cell flow in capillaries adjacent to muscle fibers (11, 15). Therefore, if mitochondrial capacity and peripheral diffusion under these conditions exert a stronger influence on V̇o_2max, a CO-induced increase in mitochondrial oxidative capacity and capillarity might be detected more easily as an increase in V̇o_2max during single-leg exercise at altitude.

Of equal interest and importance was the possibility that the induction of nuclear- and mitochondrial-encoded genes for mitochondrial proliferation and capillary O₂ delivery in skeletal muscle would be differentially expressed depending on CO dose. If HO-1/CO operates at an early regulatory step in the transcriptional programming for aerobic metabolism, a higher tissue CO level should lead to stronger upregulation of metabolic-capillary units. Moreover, an improvement in the capacity for O₂ exchange could be reflected through enhancement of O₂ availability in the microcirculation, facilitating a higher V̇o_2max on the single-leg exercise protocol. The findings indicate that inhaled CO increases muscle mitochondrial volume density without changes in V̇o_2max as seen previously but remarkably upregulates muscle myoglobin and GLUT4 content and muscle capillarity, all consistent with an unexpected regulatory role for the HO-1/CO system in skeletal muscle plasticity.

METHODS

Subjects.

After institutional review board approval and written, informed consent, 37 healthy, nonsmoking subjects were enrolled in the study (23 men and 14 women, ages 18–40 yr). Exclusion criteria were cardiovascular or respiratory illness, pregnancy, unequal leg strength, sickle trait, and altitude exposure within 24 h of study. Subjects with a very high degree of physical fitness were also excluded to eliminate any confounding effect from an already optimized V̇o_2max (and capillary density) in highly trained subjects. Subjects specifically agreed to maintain their usual level of exercise for the duration of the study. They were asked to abstain from caffeine on exercise test days and to refrain from oral analgesics prior to skeletal muscle biopsies. Three subjects could not complete the first exercise protocol and were removed from the study to make the final number 34 subjects. Each was assigned to one of four groups: exercise at simulated altitude and CO exposure (n = 11, group A or CO+E); exercise at simulated altitude and air exposure (n = 8, group B or Air + E); CO exposure only (n = 10, group C or CO); or simulated altitude exposure only (n = 5, group D or HH). Simulated altitude (hypobaric hypoxia, HH) alone was used to control any effect of hypobaric hypoxia during exposure for exercise sessions.

Muscle biopsies.

Baseline vastus lateralis (VL) muscle biopsies were performed using local anesthesia and a University College Hospital Muscle Biopsy Needle (Popper & Sons, New Hyde Park, NY; 120 mm, 5.0 mm OD). Three samples were obtained (total weight ∼200 mg) and two were snap frozen in liquid N₂ and the third immediately fixed in 10% formalin. Small 1- to 2-mm cubes were also fixed in 2% paraformaldehyde and 1% glutaraldehyde. On day 8, a second VL biopsy was performed on the contralateral leg in the same manner as the first.

Exercise protocol.

At least 24 h after each biopsy, each subject completed a staged 5-min practice session on a cycle ergometer (Monark, model 818E) in air to determine appropriate ergometer settings and to familiarize them with the apparatus. The settings established during the practice session (seat height, handlebar elevation and angle, pedal strap) and stationary foot rest (height) were held constant for each of the V̇o_2max exercise sessions.

One day later, the subjects performed a graded, single-legged V̇o_2max exercise test with the leg ipsilateral to the most recent biopsy at 15,000 ft. equivalent (pbar 429 mmHg) in a hypobaric chamber. The subjects cycled at 75 rpm and workload was increased at preset intervals. Verbal encouragement was provided to help elicit maximal effort during exercise. Exercise termination criteria were inability to maintain cadence, development of cramps, fatigue, and subject request. After exercise, the subjects remained on the apparatus for an additional 5 min for recovery measurements.

Subjects received a radial arterial line for hemodynamic monitoring before the exercise protocol (Chart software version 4.2.4) and to obtain blood samples for measurement of blood gases and lactate. These samples (3–4 ml each) were taken after baseline resting measurements and 5 min after cessation of exercise. Samples were immediately placed on ice. Blood gas measurements and CO-oximetry were performed within 20 min (Instrumentation Laboratory, GEM Premier 3000 and 682 CO-oximeter). Subjects were monitored by three-lead ECG (Spacelabs, model 511) and pulse oximeter (Ohmeda, model 3740).

Subjects wore a nose-clip and breathed air through a two-way nonrebreathing valve (Hans Rudolf, model 2700) connected to a metabolic cart (Consentius Technologies, ParvoMedics TrueMax 2400). Mixed expired oxygen (O₂), carbon dioxide (CO₂), respiratory rate, tidal volume, minute ventilation, respiratory exchange ratio, O₂ consumption, CO₂ elimination, and workload were measured with the metabolic cart and recorded every 30 s as 30-s averages. V̇o_2max was the highest O₂ uptake (V̇o₂) reached during the exercise protocol. If subjects exhibited a V̇o₂ plateau on a graph of V̇o₂ vs. workload, this was documented.

CO protocol.

The 19 subjects who underwent exercise testing (groups A and B) were assigned in a double-blind fashion to one of two groups. Group A breathed premixed CO in air (200 ppm) by mouthpiece and nose clip for 1 h/day for 5 consecutive days. Group B breathed room air using the same type of apparatus. The first gas breathing session was done later on the day of the V̇o_2max test. On day 6, immediately after the breathing session, a venous blood sample was drawn to measure the percentage of carboxyhemoglobin (COHb). Three days after the final breathing day (day 9), the second VL muscle biopsy was performed on the contralateral leg. Subjects then performed a practice session with the contralateral leg on day 10, and on day 11 they repeated the full single-legged cycling V̇o_2max exercise test with that leg (contralateral to the leg used for the first exercise test). The remaining 15 subjects served as controls and had muscle biopsies before and after either five sessions of CO breathing at sea level with no exercise (n = 10, group C), to control for effects of exercise testing in group A, or two sessions of breathing air at 15,000 feet (n = 5, group D), Po₂ 90 mmHg, as a control for the effects of hypobaric hypoxia for groups A and B. A summary of the timing of biopsies, CO/air breathing, exercise testing, and altitude exposures is provided in Table 1.

Table 1.

Study design for timing of muscle biopsies, CO exposure, and exercise testing for groups A–D

Day	Group A	Group B	Group C	Group D
0	VL muscle biopsy	VL muscle biopsy	VL muscle biopsy	VL muscle biopsy
1	Exercise practice at sea level	Exercise practice at sea level
2	Morning: Ipsilateral SL V̇o2max testing at altitude	Morning: Ipsilateral SL V̇o2max testing at altitude	Afternoon: CO 200 ppm 1 h	Altitude exposure
	Afternoon: CO 200 ppm 1 h	Afternoon: CO 200 ppm 1 h
3	CO 200 ppm 1 h	Room air 1 h	CO 200 ppm 1 h
4	CO 200 ppm 1 h	Room air 1 h	CO 200 ppm 1 h
5	CO 200 ppm 1 h	Room air 1 h	CO 200 ppm 1 h
6	CO 200 ppm 1 h	Room air 1 h	CO 200 ppm 1 h
9	Contralateral VL muscle biopsy	Contralateral VL muscle biopsy	Contralateral VL muscle biopsy	Contralateral VL muscle biopsy
10	Exercise practice at sea level	Exercise practice at sea level
11	Contralateral SL V̇o2max testing at altitude	Contralateral SL V̇o2max testing at altitude		Altitude exposure

Group A, exercise at simulated altitude and carbon monoxide (CO) exposure; group B, exercise at simulated altitude and air exposure; group C, CO exposure only; group D, simulated altitude exposure only. VL, vastus lateralus; SL, single leg; V̇o_2max, maximal O₂ uptake. Altitude = 15,000 ft. equivalent hypoxia in hypobaric chamber.

The CO exposure for these studies was set at 200 ppm for 1 h and within OSHA limits for safe environmental exposures for CO (35). The accumulation of CO from the 1-h breathing sessions would also not be significant because the sessions were separated by ∼24 h, or four to six half-lives for COHb elimination (10).

Lactate quantification.

At the time of each arterial blood draw, 1 ml was obtained for blood lactate concentrations. Enzymatic quantification of blood lactate concentrations was performed with standard fluorometric assays.

Quantitative real-time RT-PCR.

Total RNA was extracted from the muscle biopsy samples by use of TRIzol reagent (Invitrogen, Carlsbad, CA). RNA (1 μg) was reverse-transcribed by using random hexomer primers and a Superscript enzyme (Invitrogen). Real-time RT-PCR was performed with an ABI Prism 7000 and gene expression master mix (Applied Biosystems, Foster City, CA). Primers and probes from Applied Biosystems were used for human, cytochrome c oxidase subunit I (COX I), COX IV, mitofusin-1 (MFN1), mitofusin-2 (MFN2), NADH dehydrogenase subunit I (ND1), mitochondrial fission 1 (FIS1), and optic atrophy protein 1 (OPA1). 18S rRNA was used as an internal control. Differences in gene expression were quantified by using ABI Prism 7000 SDS Software. The threshold cycle (Ct) was determined in the exponential amplification phase. The amount of transcript was normalized to 18S RNA by subtracting mean Ct values for each condition. For the quantitative RT-PCR (qRT-PCR) reaction, a difference of 1.0 in Ct value represents a twofold difference in transcript level. qRT-PCR was performed in triplicate.

Quantitative PCR for mtDNA copy number.

Total and mtDNA was isolated from VL muscle specimens by use of the mtDNA Extractor Kit (Wako Chemicals, Richmond, VA). DNA primers were designed to detect COX II and 18S rDNA at a maximum amplicon length of 150 bp: 18S rDNA forward, 5′-GAATTCCCAGTAAGTGCGGGTCA-3′, and reverse, 5′-TAATGATCCTTCCGCAGGTTC-3′; COX II forward, 5′-ATGGCACATGCAGCGCAAGTAGG-3′, and reverse, 5′-ATTAGTTAGTTTTGTTGTGAGTGT-3′. The PCR reaction mixture contained 1× platinum SYBR green qPCR SuperMix UDG (Invitrogen), 500 nM of each primer, and 10 ng of total genomic DNA or mtDNA. Real-time PCR conditions were 2 min at 50°C and 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 60 s at 60°C by using the ABI Prism 7000 sequence detector system (Applied Biosystems 7000 SDS software). The Ct values in the linear exponential phase were used to measure the original DNA template copy numbers from a standard curve generated from five 10-fold dilutions of either pure mtDNA or pure nuclear 18S rDNA. Relative values for COX I and 18S rDNA within samples were used to obtain mtDNA to nuclear DNA ratios.

Western blot analysis.

Muscle proteins were separated by SDS-PAGE and identified by Western blot analysis (47). Membranes were incubated with validated polyclonal antibodies against human HO-1 (1:1,000), myoglobin (1:500), GLUT4 (1:1,000), nuclear respiratory factor 1 (NRF-1; 1:1,000), nuclear respiratory factor 2 (NRF-2 or GABPA; 1:1,000), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α, 1:500), Polγ (1:1,000), Tfam (1:1,000), mitochondrial dimethyladenosine transferase 2 (TFB2M; 1:200) (Santa Cruz Biotechnology, Dallas, TX or Invitrogen) or vascular endothelial growth factor [VEGF Ab-4 (1:500); Thermo Fisher Scientific, Waltham, MA], using β-actin staining (1:5,000; Sigma) as a loading control. After three washes in Tris-buffered saline with Tween, membranes were incubated at 1:10,000 horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham, GE Healthcare, Little Chalfont, Buckinghamshire, UK). Blots were developed with enhanced chemiluminescence, and proteins were quantified by densitometry on digitized images from the middynamic range and expressed relative to β-actin.

Immunofluorescence microscopy.

Paraffin-embedded muscle sections (4 μm) were processed for immunostaining by deparaffinization in xylene and rehydration through a descending series of alcohols, washed extensively in 0.1 M PBS, blocked with 10% normal goat serum with avidin, and incubated in primary antibody diluted in 10% normal goat serum and biotin overnight at 4°C. Primary antisera to cluster of differentiation 31 (CD31; PECAM-1), Slow Skeletal Myosin Heavy chain (myosin-7; Abcam), citrate synthase (CS; Invitrogen), or GLUT4 (Abcam) were used at a working dilution of 1:100. To detect CD31, myosin-7, CS and GLUT4, sections were washed extensively in PBS and incubated in IgG secondary labeled with Alexa Fluor 488 green or Alexa Fluor 556 red. Nuclei were counterstained with DAPI (blue). Images were acquired on a Nikon E400 fluorescence microscope (Nikon, Tokyo, Japan). Digitally captured images were analyzed with NIS-Elements imaging software (Nikon); fluorescence was quantified in eight fields per section, and number per fiber of CD31-positive vessels was quantified. Endothelial cells were counted in eight random moderate-power (×40) fields from each biopsy and expressed as the number of CD31-positive cells per muscle fiber for each field.

Electron microscopy.

The 1- to 2-mm cubes of VL muscle fixed with 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M PBS (pH 7.4) were blocked and fixed in 1% osmium tetroxide aqueous solution (Sigma-Aldrich) for 1 h, washed for 10 min three times with double-distilled H₂O, then dehydrated in graded ethanol and propylene oxide (Sigma-Aldrich). The samples were embedded in Epon at room temperature and polymerized at 55°C for 1 day. Ultrathin sections (60 nm) were cut, contrasted with uranyl acetate followed by lead citrate and examined by transmission electron microscopy (TEM) (Phillips CM12). Mitochondrial numbers were counted in five fields, with three grids per group; mitochondrial volume density was calculated by standard stereological methods (3, 51).

Statistics.

Grouped data are reported as means ± SE. Experiment-wide two-way ANOVAs were run to test normality and equal variance, and both were passed. Student's t-tests were used to evaluate pre- vs. postbiopsy measurements for each group. Values of P < 0.05 were considered significant and except when indicated; no multiple-comparison data are reported.

RESULTS

Study population.

The demographic characteristics of the 34 subjects in all four groups are provided in Table 2. There were no significant differences in age or baseline values. Of the 20 subjects assigned to groups A and B, nine could not be included in the final analysis of the V̇o_2max tests. Two were excluded for technical equipment malfunctions. Four ceased exercise prematurely for reasons other than fatigue [cramps (n = 2), shortness of breath (n = 1), and feeling ill (n = 1)]. One subject dropped out of the study and two were excluded because of significant variation between the exercise performances that were attributable to extraneous factors. Seven subjects in group A (CO+Ex) and four subjects in group B (Air+Ex) were included in the final exercise test analysis. Group C received CO only (n = 10) and group D was exposed to altitude alone (HH n = 5). Baseline COHb values were the same for each group [group A 1.5 ± 0.25%; group B 1.5 ± 0.26%; all unexercised controls (groups C and D) 1.3 ± 0.29%]. Compared with preexposure baseline and to air breathing controls, the CO exposure (200 ppm for 1 h/day for 5 days) increased mean COHb level (group A 5.4 ± 0.79%; group C 5.6 ± 0.36%; P < 0.05 pre-CO vs. after 5th day of CO).

Table 2.

Demographics and baseline measurements

Demographics and Baseline Measurements							Preexposure Values		Post-CO Exposure
Group	Conditions	V̇o2max test	n	Mean age, yr (range)	Male	Female	COHb% (means ± SD)	V̇o2peak, l/min) (means ± SD)	COHb% (means ± SD)
A	CO 200 ppm 1 h × 5 days	yes	11	23.1 (20–27)	8	3	1.46 ± 0.25	1.86 ± 0.51	5.40 ± 0.79
B	Room air 1 h × 5 days	yes	8	24.1 (21–27)	6	2	1.50 ± 0.26	1.78 ± 0.34
C	CO 200 ppm 1 h × 5 days	no	10	26.2 (21–40)	5	5	1.28 ± 0.29		5.60 ± 0.36
D	Hypobaric hypoxia control	no	5	25.2 (18–34)	3	2
Failed			3	27.0 (21–37)	1	2
Totals			37		23	14

Biochemical and molecular data are reported for the four subject groups in Table 2. No cross-group comparisons from experiment-wide ANOVAs are reported except that no post-CO comparisons for groups A vs. C were significant, whereas post-CO (A or C) vs. post-air (B) signals were significant in Figs. 1–4 below.

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. 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.

CO and mitochondrial biogenesis.

Muscle HO-1 protein levels determined by Western blot analysis showed consistent increases only in the subjects who breathed CO, with or without exercise [groups A (CO+E) and C (CO)]. By densitometry, normalized to β-actin, mean HO-1 protein levels increased by more than twofold in group A and approximately twofold in group C (P < 0.05) but did not change in air-breathing exercise controls (group B) or HH control subjects (group D; Fig. 1A). In addition, there was a statistically significant increase in mtDNA copy number (∼40%) after 5 days of CO breathing in exercised and nonexercised subjects (groups A and C), but not in the air or altitude protocol groups (Fig. 1B). There was no difference between the two CO groups with or without exercise testing; thus one bout of single-legged exercise did not significantly alter mtDNA copy number in the contralateral leg.

There were significant increases in VL muscle protein content for the transcriptional activators NRF-1, GABPA, and the PGC-1α coactivator by Western blot analysis in the CO-exposed subjects in groups A and C (P < 0.05; Fig. 2). By densitometry, NRF-1 increased by 50–70% after CO while PGC-1α levels doubled. There was no significant change in air-breathing subjects or in altitude controls (groups B and D).

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.

The transcription and replication of mtDNA are regulated by a group of nuclear-encoded proteins of which Polγ, Tfam, and Tfb2m are representative. The levels of these three proteins were increased by up to twofold in VL muscle after CO breathing; this was also independent of exercise testing (Fig. 3) but did not change in air-breathing or in altitude controls.

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.

The mRNA levels for two nuclear-encoded and two mitochondrial-encoded transcripts were evaluated by qRT-PCR (Fig. 4). There were significant increases in both the nuclear-encoded transcripts (COX IV, cytochrome c) and in the mtDNA-encoded transcripts (ND1, COX I) in the CO breathing groups (groups A and C), but not in the air or altitude groups (groups B and D). Hence, these genes are highly sensitive to activation by HO-1/CO in human skeletal muscle.

Muscle mitochondrial density and phenotype.

Changes in muscle mitochondrial density were evaluated both by immunofluorescence staining for CS and by TEM. CS immunofluorescence of VL sections for representative subjects in each group pre- and postintervention is shown in Fig. 5A. CS fluorescence is semiquantitative, but it effectively detects differences in mitochondrial content. Significant changes occurred in the CO-exposed groups, but among those muscle sections only ∼70% of the fibers demonstrated an increase in CS fluorescence. The sections were also stained for myosin-7, which indicated that CS upregulation occurred mainly in fibers already committed to an oxidative phenotype (Fig. 5B). We found no evidence of phenotype switching.

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.

The increase in muscle mitochondrial volume density evident after CO was confirmed by TEM (Fig. 6). Fig. 6A shows representative electron photomicrographs, and Fig. 6B shows the analysis of the number of mitochondrial profiles in five randomly selected fields of each biopsy for the four groups [group A 142.0 ± 27.8 pre-CO, 235.0 ± 36.5 post-CO, P = 0.002; group B 138.0 ± 27.8 pre-air, 155.0 ± 35.0 post-air, P = not significant (NS); group C 139.0 ± 34.9 pre-CO, 219.0 ± 36.2 post-CO, P = 0.007; group D 158.0 ± 25.1 pre-altitude, 178.0 ± 25.7 post-altitude, P = NS; no. of mitochondrial profiles/field]. Qualitatively, we observed that CO led to densely packed accumulations of subsarcolemmal mitochondria especially adjacent to capillaries and around nuclei. The content of intermyofibrillar mitochondria also increased after CO, and these were arranged in a closer spatially organized pattern compared with control muscle.

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.

Gene expression for mitochondrial dynamics.

To assess the subcellular distribution of mitochondria in relation to changes in the expression of genes involved in mitochondrial dynamics, mRNA levels of mitochondrial fission and fusion proteins MFN1, MFN2, FIS1, and OPA1 were measured in the VL biopsies (Fig. 7). No differences were found in the levels of muscle mRNA for FIS1 and MFN1; however, MFN2 and OPA1 increased significantly in the CO breathing subjects, but not in the air breathing or HH controls.

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.

Glucose transport.

Because changes in O₂ and substrate availability are needed to support energy metabolism, we checked for evidence of altered glucose availability by measuring GLUT4 expression levels and localization in the myocytes. Breathing CO for 5 days increased GLUT4 protein expression by approximately twofold (Fig. 8A). By immunofluorescence microscopy, we also determined the subcellular distribution of GLUT4 and found GLUT4 localized in air and altitude control subjects mainly in the cytoplasm of myocytes with a few labeled punctate structures in the cell periphery (Fig. 8, B and C). In contrast, the CO-exposed subjects demonstrated substantial redistribution of GLUT4 to the plasma membrane. There was also a modest redistribution in the altitude control group, consistent with prior reports (6). In addition, we found that CO had affected the intracellular content of myoglobin in the muscle. CO breathing, irrespective of exercise, caused a significant increase in myoglobin protein (Fig. 8D). Moreover, there was no change in myoglobin content after comparable altitude exposure, indicating that this effect was not attributable to tissue hypoxia during CO breathing.

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.

A semiquantitative analysis of muscle capillarity was performed. VL sections from air and CO breathing subjects were immunostained with antibodies against the endothelial-specific capillary marker CD31. The average number of fibers counted was 179 per group. The number of CD31-positive capillaries per muscle fiber (group A 1.78 ± 0.15 pre-CO, 2.37 ± 0.59 post-CO, P = 0.01; group B 1.79 ± 0.32 pre-air, 1.90 ± 0.39 post-air, P = 0.36; group C 1.93 ± 0.30 pre-CO, 2.46 ± 0.73 post-CO, P = 0.03; group D 1.81 ± 0.21 pre-altitude, 2.30 ± 0.28 post-altitude, P = 0.11; capillaries/muscle fiber) increased significantly in groups A and C after CO breathing, supporting slightly higher muscle capillarity and improved muscle nutrient perfusion (Fig. 9). Skeletal muscle VEGF protein levels also increased, consistent with increased capillarization (Fig. 9C) (group A 0.78 ± 0.21 pre-CO, 1.42 ± 0.23 post-CO, P < 0.01; group B 0.79 ± 0.08 pre-air, 0.93 ± 0.18 post-air, P = 0.28; group C 0.93 ± 0.18 pre-CO, 1.21 ± 0.29 post-CO, P = 0.05; group D 0.80 ± 0.16 pre-altitude, 1.14 ± 0.25 post-altitude, P = 0.04; VEGF/actin) and are also increased in the HH group, consistent with regulation of VEGF expression by hypoxia (26). The hypoxic stimulus was not repeated as was the CO exposure, which may explain the observed difference in VEGF in group D (HH only) without a significant increase in capillarity, as demonstrated in groups A and C (CO).

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.

Exercise testing.

Seven subjects (4 group A; 3 group B) reached a traditional V̇o_2max on both exercise runs, demonstrating a plateau in V̇o₂ on the graph of V̇o₂ vs. workload (Fig. 10). There was no significant change in V̇o_2max from baseline in subjects exposed to CO and no difference compared with air breathing controls (group A: 1.96 ± 0.51 pre-CO, 1.87 ± 0.50 post-CO l/min; group B: 1.90 ± 0.43 pre-air, 1.90 ± 0.47 post-air l/min). Mean hemodynamic and respiratory values after completion of the exercise protocol are provided for all subjects (groups A and B) in Table 3. Both groups had similar baseline values and there were no significant differences after either protocol.

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 V̇o_2max.

Table 3.

Exercise data for subjects completing both V̇o_2max exercise tests at altitude before and after exposures

	N	V̇o2max, l/min STPD	V̇o2, ml·kg−1·min−1 STPD	V̇o2max % change	METS	Max HR	Max V̇e, l/min BTPS	Max RER	V̇o2, l/min STPD	Max load, W	Max SBP
Group A CO pre	7	1.96 ± 0.51	24.79 ± 5.52	−4.3 ± 9.7	7.08 ± 1.58	181.5 ± 10.7	105.7 ± 28.2	1.31 ± 0.15	2.38 ± 0.50	80.6 ± 43.1	193 ± 19.5
Group A CO post	7	1.87 ± 0.5	23.69 ± 5.77		6.77 ± 1.65	178.5 ± 9.3	106.7 ± 31.1	1.34 ± 0.12	2.33 ± 0.47	74.6 ± 47.4	198.6 ± 23.7
Group B Control pre	4	1.9 ± 0.43	23.86 ± 4.21	−0.3 ± 7.8		172.7 ± 19.7	105.1 ± 23.2		2.4 ± 0.60	73.4 ± 24.1	187.1 ± 8.0
					6.82 ± 1.20			1.34 ± 0.16
Group B Control post	4	1.9 ± 0.47	23.72 ± 3.83		6.78 ± 1.09	173.9 ± 13.1	101.2 ± 18.9		2.3 ± 0.64	74.9 ± 27.8	185.8 ± 22.3
								1.24 ± 0.08

Data are means ± SD. METS, metabolic equivalents; HR, heart rate; V̇e, minute ventilation; RER, respiratory exchange ratio; SBP, systolic blood pressure; Max, maximal. Group A: CO 200 ppm 1 h × 5 days; group B: room air 1 h × 5 days.

Arterial blood gas, pH, and % oxyhemoglobin measurements for both groups are shown in Table 4. Changes with exercise were all significant in both groups (P < 0.05) but were not different between the two groups. Blood lactate concentrations were similar for CO and air breathing subjects at baseline and at 5 min after the first V̇o_2max test (10.6 ± 2.3 and 10.6 ± 4.3 mmol/l, respectively; P = NS).

Table 4.

Arterial blood gas values and blood lactate concentrations for V̇o_2max exercise testing at altitude

		pH			PaCO2, mmHg			PaCO2, mmHg			Blood Lactate, mmol/l			O2Hb%
	N	Rest	Stop	Final	Rest	Stop	Final	Rest	Stop	Final	Rest	Stop	Final	Rest	Stop	Final
Group A CO pre	7	7.46 ± 0.03	7.33 ± 0.05	7.28 ± 0.04	33.4 ± 2.9	26.7 ± 5.8	26.4 ± 3.6	38.9 ± 3.9	43.0 ± 4.2	49.0 ± 2.4	0.9 ± 0.7	11.1 ± 5.9	10.6 ± 2.3	77 ± 4.0	77.3* ± 4.8	81.3 ± 3.1
Group A CO post	7	7.46 ± 0.05	7.32 ± 0.06	7.26 ± 0.02	33.4 ± 4.6	26.9 ± 4.6	26.3 ± 2.9	42.9 ± 7.4	40.6 ± 5.8	51.4 ± 2.4	0.9 ± 0.4	11.5 ± 4.4	11.9 ± 2.1	80 ± 7.1	72.7* ± 8.7	81.7 ± 2.5
Group B Control pre	4	7.47 ± 0.05	7.32 ± 0.07	7.29 ± 0.09	31.8 ± 5.1	24.8 ± 4.4	24.8 ± 3.1	42 ± 7.5	40.8 ± 4.0	50.8 ± 5.7	0.7 ± 0.3	10.1 ± 4.0	10.6 ± 4.3	82.8 ± 6.5	75 ± 7.1	82.8 ± 6.5
Group B Control post	4	7.46 ± 0.03	7.35 ± 0.05	7.28 ± 0.08	33.0 ± 5.0	25.0 ± 4.1	25.5 ± 1.7	42.5 ± 7.1	41.5 ± 4.0	49.5 ± 3.1	0.5 ± 0.2	11.9 ± 4.3	11.7 ± 5.2	80.4 ± 7.3	76.4 ± 5.8	82.2 ± 4.3

Data are means ± SD. Rest, before exercising; Stop, immediately after exercise. Final = 5 min after exercise.

^*P < 0.05.

DISCUSSION

The present study confirms and extends previous work from our laboratory on the induction of mitochondrial biogenesis by CO and provides novel insights into the role of the HO-1/CO system in muscle adaptation. We report not only an increase in mtDNA copy number, but also four other novel findings including increases in myocyte membrane GLUT4 transporter levels and intracellular myoglobin content, remodeling of the mitochondrial network by enhancement of fusion-protein gene expression, and stimulation of muscle capillarity.

Mitochondrial quality control responses to inhaled CO were assessed at tissue levels of CO close to those found upon induction of the HO-1/CO system (36). All subjects had normal pulmonary gas exchange and those receiving CO breathed the gas at 200 ppm for 1 h/day for 5 consecutive days. These exposures were safely performed to peak COHb levels of ∼6% without reported side effects. Our CO protocol was based on information that 100 ppm CO could be given safely for 1 h/day for 5 consecutive days but only partially induced the mitochondrial biogenesis program in VL muscle in healthy subjects despite HO-1 induction (43).

In both human studies, the molecular responses to CO did not impact the measured V̇o_2max; however, neither used physical training in conjunction with CO. Thus normal training-related changes in contractile function, muscle mass (23), or blood flow that also contribute to increases in V̇o_2max may not have occurred. However, muscle oxidative capacity is also linked to overall metabolic health (4, 45); hence, the CO-mediated increases in muscle mitochondrial volume density may have an advantage that is unrelated to V̇o_2max. The effects of CO at the higher dose in the examination of a larger group of mitochondrial quality control regulators revealed several important new effects. We confirmed that CO breathing induced the HO-1/CO system along with the NRF-1 and NRF2 transcription factors and the PGC-1α coactivator, all crucial for mitochondrial gene transcription (46), but newly demonstrate that the higher CO dose led to further activation of the program as shown by an increase in muscle mtDNA content, TCA cycle enzymes, and mitochondrial fusion proteins.

The VL muscle is useful for mitochondrial studies because of its accessibility, but also because it is comprised mainly of oxidative fibers. The human VL contains ∼40% slow oxidative fibers, 30% fast oxidative fibers, and 30% various fast glycolytic fibers (14). In our previous study (43), and in the fiber composition of subjects here, we found no effect of CO on type I fiber composition, indicating that mitochondrial proliferation was occurring mainly in existing oxidative fibers and not because of fiber type switching. This also implies that the observed mitochondrial responses represent the selection of specific molecular characteristics needed to effect a change in mitochondrial phenotype (39). Such changes may include differences in substrate preference, regulation of oxidative phosphorylation, Ca²⁺ handling, or reactive oxygen species signaling.

The mitochondrial volume density of muscle is increased by training, especially with acute endurance (40) and interval training (13). An early and essential factor is PGC-1α, and, in mice, muscle mitochondrial content and the capacity for endurance exercise increase with muscle-specific PGC-1α overexpression (5). Similarly, we observed PGC-1α responses with CO exposure in the absence of training along with those of other markers of mitochondrial biogenesis, Polγ, Tfam, and TFB2M that are also integral to mitochondrial biogenesis. Accordingly, the induction of mitochondrial biogenesis after CO is supported by increased mRNA levels for nuclear-encoded cytochrome c and COX I and for mitochondrial-encoded ND1 and COX IV, together indicating that mtDNA replication and transcription and mitochondrial protein synthesis are highly sensitive to tissue CO content. By comparison, in muscle cells, interference with the HO-1/CO system by gene silencing abrogates the induction of PGC-1α and mitochondrial biogenesis by CO (38). Further corroboration was found here in the CO-mediated increase in protein for nuclear-encoded TCA cycle CS, which correlates with mitochondrial volume density. Also, we found enhanced genetic signatures for proteins that have roles in the construction of dynamic mitochondrial networks, specifically the OPA1 cristal protein and the MFN2 fusion protein. The redox-dependent effects of CO stimulate cellular mitochondrial content in many tissues, and although mitochondrial H₂O₂ release is required (38), the cell regulation is not fully understood. An increase in mitochondrial functional reserve, that is an improved bioenergetic capacity, requires not only the coordinated expression of nuclear and mitochondrial-encoded genes that regulate mitochondrial biogenesis, but proteins that regulate the ancillary pathways critical for substrate and O₂ provision. In this context, the expression of myoglobin, the main facilitator of O₂ transfer from cell membrane to mitochondria (16), is increased by CO exposure. Moreover, increases in VEGF and number of CD31-positive capillaries per myofiber were found in CO-treated subjects, which would presumably support an increased oxidative capacity.

It is reported that the HO-1/CO system improves both insulin release and sensitivity (24, 27, 34). Although we did not measure insulin responsiveness, mitochondrial biogenesis is associated with improved oxidative phosphorylation in muscle, which is a key determinant of insulin sensitivity. The increase in muscle GLUT4 expression and its membrane localization after CO support the link between oxidative capacity and insulin sensitivity and articulates somatic energy regulation by skeletal muscle mitochondria (18). This may indicate an improvement in insulin sensitivity, since membrane GLUT4 levels in muscle correlate with a higher glucose transport capacity (30).

Insulin recruits GLUT4 from intracellular sites to the cell membrane, where it mediates insulin- and contraction-stimulated transport of glucose into cells (20). GLUT4 gene expression is mediated by MEF2A/2D transcription factors and GLUT4 enhancer factor (GEF) (31, 32), whereas PGC-1a coactivates MEF2A (19) and, by activating NRF-1, increases MEF2A protein expression (2, 41). GLUT4 rapidly responds to exercise (7, 17), but the protein's short half-life contributes to a rapid reversibility of GLUT4 translocation (12). In rat skeletal muscle after a single bout of exercise, GLUT4 upregulation results in significant increases in glucose transport and muscle glycogen synthesis (42). Moreover, PGC-1α activates the estrogen-related receptor α (ERRα) transcription factor that regulates PDK expression, which also mediates the insulin response. PDK phosphorylates pyruvate dehydrogenase (PDH), decreasing its activity and limiting pyruvate entry into the TCA cycle. Glucose oxidation rates therefore fall, whereas fatty acid metabolism rises. Functional measurements of glucose transport or glucose oxidation are needed to support these suppositions, but the transcriptional activation of mitochondrial biogenesis by CO supports the possible enhancement of mitochondrial oxidative capacity and insulin-mediated glucose transport through GLUT4.

In summary, CO breathing in humans activates mitochondrial biogenesis in VL muscle with no measurable increase in V̇o_2max, but with accompanying increases in muscle myoglobin and GLUT4 content as well as in muscle capillarity. Capillary-oxidative metabolic units in muscle are responding to tissue CO independently of tissue hypoxia, which has more limited effects. Although added capacities for nutrient perfusion and muscle performance were not demonstrable by increases in V̇o_2max, a lack of performance improvement relative to plasticity is not unexpected given the absence of training. Thus CO alone is insufficient to increase V̇o_2max, but HO-1/CO can be implicated in functional regulation beyond the parameters in this study, such as insulin sensitivity, or in setting the work capacity in endurance training. The HO-1/CO-induced mitochondrial changes in this study may manifest as changes in substrate preference, and therefore in efficiency at a given workload during steady-state exercise, rather than as an increase in V̇o_2max. Though such ergogenic implications remain untested, it is clear that exogenous CO pharmacologically activates mitochondrial quality control pathways, and that manipulation of oxidative plasticity in human muscle through inhalation of the gas is feasible.

GRANTS

Supported by National Heart, Lung, and Blood Institute Grants R01 HL090679 and P01 HL108801.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

S.R.H.P., J.V.P., A.D.C., D.F.P., R.E.M., and H.B.S. performed experiments; S.R.H.P., J.V.P., A.D.C., D.F.P., K.E.W.-W., R.E.M., C.A.P., and H.B.S. analyzed data; S.R.H.P., J.V.P., A.D.C., K.E.W.-W., R.E.M., C.A.P., and H.B.S. interpreted results of experiments; S.R.H.P. drafted manuscript; S.R.H.P., J.V.P., A.D.C., D.F.P., K.E.W.-W., R.E.M., C.A.P., and H.B.S. approved final version of manuscript; A.D.C., C.A.P., and H.B.S. prepared figures; A.D.C., R.E.M., C.A.P., and H.B.S. edited and revised manuscript; R.E.M. and C.A.P. conception and design of research.