Skeletal muscle knockout of NAD(P)H oxidase 2 delays the development of isotonic diaphragm fatigue in mice.

Mechanisms of skeletal muscle fatigue are commonly studied under isometric conditions, which exclude muscle shortening and limit physiological relevance. We developed a novel in vitro protocol to examine isotonic fatigue using afterload contractions that permits the study of additional active (velocity, power, work) and passive (stiffness, energy loss) mechanical properties of muscle. During the development of this protocol, we examined the impact of shortening load during afterload contractions on the development of fatigue, and observed a relationship where fatigue onset is more rapid and severe with larger shortening loads (30 % vs. 45 % vs. 60 % maximal isometric force). We then applied this protocol to investigate the contribution of NAD(P)H Oxidase 2 (Nox2) to fatigue development and recovery. Nox2 was deleted from skeletal muscle using the Cre-LoxP system (skmNox2KO), while Cre-negative littermates were used as controls. Knockout of Nox2 attenuated the decline in power and increased total isotonic work performed during repeated contractions compared to controls. Recovery kinetics of power, work, and isometric force were similar between groups. Passive mechanical properties-including stiffness and energy loss- increased with fatigue but were unaffected by Nox2 deletion. These findings highlight the importance of incorporating isotonic contractions to uncover fatigue mechanisms and suggest that Nox2, and presumably reactive oxygen species, contributes to the decline in muscle power during repetitive shortening contractions.