Studies of mitochondrial and nonmitochondrial sources implicate nicotinamide adenine dinucleotide phosphate oxidase(s) in the increased skeletal muscle superoxide generation that occurs during contractile activity.
OBJECTIVE
The sources of cytosolic superoxide in skeletal muscle have not been defined. This study examined the subcellular sites that contribute to cytosolic superoxide in mature single muscle fibers at rest and during contractile activity.


RESULTS
Isolated fibers from mouse flexor digitorum brevis loaded with superoxide and nitric-oxide-sensitive fluorescent probes, specific pathway inhibitors and immunolocalization techniques were used to identify subcellular sites contributing to cytosolic superoxide. Treatment with the electron transport chain complex III inhibitor, antimycin A, but not the complex I inhibitor, rotenone, caused increased cytosolic superoxide through release from the mitochondrial intermembrane space via voltage-dependent anion or Bax channels, but inhibition of these channels did not affect contraction-induced increases in cytosolic superoxide. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors decreased cytosolic superoxide at rest and following contractions. Protein and mRNA expression of NADPH oxidase subunits was demonstrated in single fibers. NOX2, NOX4, and p22(phox) subunits localized to the sarcolemma and transverse tubules; NOX4 was additionally expressed in mitochondria. Regulatory p40(phox) and p67(phox) proteins were found in the cytoplasm of resting fibers, but following contractions, p40(phox) appeared to translocate to the sarcolemma.


METHODS
Superoxide and other reactive oxygen species generated by skeletal muscle are important regulators of muscle force production and adaptations to contractions. This study has defined the relative contribution of mitochondrial and cytosolic sources of superoxide within the cytosol of single muscle fibers at rest and during contractions.


CONCLUSIONS
Muscle mitochondria do not modulate cytosolic superoxide in skeletal muscle but NADPH oxidase is a major contributor both at rest and during contractions.
Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom.
Giorgos Konstantinos
GK
Sakellariou
Aphrodite
A
Vasilaki
Jesus
J
Palomero
Anna
A
Kayani
Lea
L
Zibrik
Anne
A
McArdle
Malcolm J
MJ
Jackson
D000818:Animals; D003573:Cytochrome b Group / Q000378:metabolism; D003600:Cytosol; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism / Q000502:physiology; D009119:Muscle Contraction / Q000235:genetics / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000378:metabolism / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism / Q000502:physiology; D009249:NADP / Q000378:metabolism; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism*; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D013481:Superoxides / Q000378:metabolism
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D013481:Superoxides; D009249:NADP; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; C515614:Cyba protein, mouse