Carbon monoxide, skeletal muscle oxidative stress, and mitochondrial biogenesis in humans

Abstract

Given that the physiology of heme oxygenase-1 (HO-1) encompasses mitochondrial biogenesis, we tested the hypothesis that the HO-1 product, carbon monoxide (CO), activates mitochondrial biogenesis in skeletal muscle and enhances maximal oxygen uptake (Vo(2max)) in humans. In 10 healthy subjects, we biopsied the vastus lateralis and performed Vo(2max) tests followed by blinded randomization to air or CO breathing (1 h/day at 100 parts/million for 5 days), a contralateral muscle biopsy on day 5, and repeat Vo(2max) testing on day 8. Six independent subjects underwent CO breathing and two muscle biopsies without exercise testing. Molecular studies were performed by real-time RT-PCR, Western blot analysis, and immunochemistry. After Vo(2max) testing plus CO breathing, significant increases were found in mRNA levels for nuclear respiratory factor-1, peroxisome proliferator-activated receptor-gamma coactivator-1alpha, mitochondrial transcription factor-A (Tfam), and DNA polymerase gamma (Polgamma) with no change in mitochondrial DNA (mtDNA) copy number or Vo(2max). Levels of myosin heavy chain I and nuclear-encoded HO-1, superoxide dismutase-2, citrate synthase, mitofusin-1 and -2, and mitochondrial-encoded cytochrome oxidase subunit-I (COX-I) and ATPase-6 proteins increased significantly. None of these responses were reproduced by Vo(2max) testing alone, whereas CO alone increased Tfam and Polgamma mRNA, and COX-I, ATPase-6, mitofusin-2, HO-1, and superoxide dismutase protein. These findings provide evidence linking the HO/CO response involved in mitochondrial biogenesis in rodents to skeletal muscle in humans through a set of responses involving regulation of the mtDNA transcriptosome and mitochondrial fusion proteins autonomously of changes in exercise capacity.

Fig. 1.

Carbon monoxide (CO) and bigenomic transcriptional activity in human skeletal muscle. Real-time RT-PCR was performed on muscle biopsy samples of healthy subjects before and after 5 days of inhaled CO or air exposure and a maximal oxygen uptake (V̇o_2max) test, or before and after 5 days inhaled CO without exercise (Ex). Relative levels of mRNA for nuclear-encoded peroxisome proliferator-activated receptor-γ coactivator (PGC-1α) (A), nuclear respiratory factor (NRF)-1 (B), mitochondrial-encoded transcription factor-A (Tfam; C) and DNA polymerase γ (Polγ) (D) are shown before and after CO with or without a V̇o_2max test. Values are means ± SD (*P < 0.05 for between-group comparisons).

Fig. 2.

Nuclear and mitochondrial protein expression and mitochondrial DNA (mtDNA) copy number. A: expression of skeletal muscle citrate synthase determined by Western blot before and after CO with or without V̇o_2max testing compared with porin, the loading control. B: mtDNA copy number in muscle before and after 5 daily exposures to air or CO and one V̇o_2max test, or before and after CO without exercise. C and D: mitochondrial-encoded cytochrome oxidase (COX)-I (C) and ATPase-6 protein (D) relative to porin before and after CO with or without V̇o_2max test. Values are means ± SD (*P < 0.05 between groups).

Fig. 3.

Immunofluorescence microscopy of skeletal muscle. COX-I (green fluorescence-mitochondrial encoding) and succinate dehydrogenase (SDH) subunit A (red fluorescence-nuclear encoding) were stained with specific antibodies, and the images were merged to assess colocalization. A–C: typical SDH (A) and COX-I (B) staining of control muscle and the overlay (C). D–F: typical stained muscle sections for SDH (D), COX-I (E), and the overlay (F) from the air plus V̇o_2max group. G–I: muscle sections from the CO plus V̇o_2max group stained for SDH (G), COX-I (H), and the overlay (I). J–K: SDH (J) and COX-I (L) staining and the overlay (K) in a sample of muscle after 5 days of CO breathing.

Fig. 4.

Oxidative stress response and protein kinase B (Akt) activation in skeletal muscle after CO breathing with or without V̇o_2max testing. Western blots for heme oxygenase (HO)-1, superoxide dismutase (SOD)-2, and phospho- and total Akt were performed on muscle samples before and after 5 days of inhaled CO, with or without the V̇o_2max test. Relative protein expression was compared by normalizing HO-1 and SOD2 to β-actin. Akt activation was determined by the ratio of phospho- to total Akt. A: representative Western blots for HO-1 and SOD-2. B: densitometry for HO-1 expression relative to β-actin. C: densitometry for SOD-2 expression relative to β-actin. D: Akt activation before and after CO exposure. Values are means ± SD (*P < 0.05 between groups).

Fig. 5.

Mitochondrial fusion [mitofusin (mFn)] proteins and optic atrophy (OPA)-1 responses to inhaled CO with or without V̇o_2max testing. A: Western blots of muscle tissue for mFn1, mFn2, and OPA-1 before and 5 days after inhaled CO with or without the V̇o_2max test. Relative protein expression was derived by normalizing mFn1, mFn2, and OPA-1 to porin, a stable mitochondrial outer membrane protein. B: relative mFn1 expression. C: relative mFn2 expression. D: relative OPA-1 expression. Values are means ± SD (*P < 0.05 between groups).

Fig. 6.

Myosin heavy chain (MHC) protein expression in skeletal muscle after CO breathing with or without V̇o_2max testing. Gels of muscle samples for MHC isoforms from subjects before and 5 days after inhaled CO with or without the V̇o_2max test. MHC I was expressed relative to β-actin, used as a loading control. Values are means ± SD (*P < 0.05 between groups).