Sarcolemma-localized nNOS is required to maintain activity after mild exercise

Abstract

Many neuromuscular conditions are characterized by an exaggerated exercise-induced fatigue response that is disproportionate to activity level. This fatigue is not necessarily correlated with greater central or peripheral fatigue in patients, and some patients experience severe fatigue without any demonstrable somatic disease. Except in myopathies that are due to specific metabolic defects, the mechanism underlying this type of fatigue remains unknown. With no treatment available, this form of inactivity is a major determinant of disability. Here we show, using mouse models, that this exaggerated fatigue response is distinct from a loss in specific force production by muscle, and that sarcolemma-localized signalling by neuronal nitric oxide synthase (nNOS) in skeletal muscle is required to maintain activity after mild exercise. We show that nNOS-null mice do not have muscle pathology and have no loss of muscle-specific force after exercise but do display this exaggerated fatigue response to mild exercise. In mouse models of nNOS mislocalization from the sarcolemma, prolonged inactivity was only relieved by pharmacologically enhancing the cGMP signal that results from muscle nNOS activation during the nitric oxide signalling response to mild exercise. Our findings suggest that the mechanism underlying the exaggerated fatigue response to mild exercise is a lack of contraction-induced signalling from sarcolemma-localized nNOS, which decreases cGMP-mediated vasomodulation in the vessels that supply active muscle after mild exercise. Sarcolemmal nNOS staining was decreased in patient biopsies from a large number of distinct myopathies, suggesting a common mechanism of fatigue. Our results suggest that patients with an exaggerated fatigue response to mild exercise would show clinical improvement in response to treatment strategies aimed at improving exercise-induced signalling.

Figures

Figure 1. Loss of sarcolemma-localized nNOS leads to skeletal muscle vascular narrowings, reduced capillary perfusion, and an exaggerated fatigue response after mild exercise in dystrophic and non-dystrophic mouse models

a, Representative C57BL/10 and mdx mice pre- and post-exercise zone map vertical activity tracings. b, Quantified vertical activity pre- and post-exercise for C57BL/10, C57BL/6, mdx and Sgca-null mouse strains (n=6 for each strain). c, EDL muscle specific force measurements from C57BL/6 (n=6), mdx (n=4), and Sgca-null (n=4) mice. d, Pre- and post-exercise vertical activity in untreated (n=7) and anti-inflammatory treated mdx mice, acutely (n=4) or chronically (n=4) with deflazcort or acutely with ibuprofen (n=5). Asterisks indicate statistical significance. e, Quantified pre and post-exercise vertical activity for Microdystrophin/mdx mice (n=6) and their mdx littermates (n=4). Insets show representative immunofluorescence images of nNOS detection in the gastrocnemius muscles from C57BL/6 and microdystrophin/mdx mice. f, Quantified pre and post-exercise vertical activity for MCKεSG/Sgca-null mice (n=6) and their Sgca-null littermates (n=6). Inset shows immunoblot detection of total nNOS from homogenates (hom), and crude skeletal muscle membranes (cm). g, Representative Microfil® image of skeletal muscle vessels of MCKεSG/Sgca-null mice post-exercise – large arrow heads mark extended areas of vascular narrowing and small arrows mark shorter stretches of radial vascular narrowing.

Figure 2. Enhancing the cGMP signal resulting from muscle nNOS activation decreases the exaggerated fatigue response to mild exercise

a, Comparison of vertical activity post-exercise among C57BL/6, eNOS- and nNOS-null mice (n=6 for each). b, Serum creatine kinase levels pre- and post-exercise in C57BL/6 (n=6), eNOS-null (n=4), and nNOS-null (n=6) mice, compared to mdx (n=6). c, Representative Microfil® image of nNOS-null quadriceps skeletal muscle arteries post-exercise – large arrow heads mark extended area of vascular narrowing and small arrows mark shorter areas of radial vascular narrowing. d, Pre- and post-exercise vertical activities in untreated wild-type (C57BL/6 and C57BL/10) mice (n=4), compared to 3-B-7-Ni treated wild-type mice (n=3), and to sarafotoxin treated wild-type mice (n=4). e, Quantified pre- and post-exercise activity +/−PDE5A inhibitor treatment, in nNOS-null (n=4), MCKεSG/Sgca-null (n=4), and mdx mice (n=6). Pre- and post-exercise vertical activity error bars are s.e.m., scale bar = 100 μm, and asterisks indicate statistical significance.

Figure 3. PDE5A inhibitor treatment improves exercised-induced vasomodulation and reduces exercise-induced edema in mdx mice

a, Representative images of coronal Laser Doppler analysis of blood flow from mdx mice pre- and post-exercise (n=3), and b, mdx mice pre- and post-exercise + PDE5A inhibitor treatment (n=3). c, Representative Microfil® image of mdx + PDE5A inhibitor treatment quadriceps skeletal muscle arteries post-exercise (n=3, Scale bar = 100 μm). d-e, Representative MRI axial views of: d, mdx hind-limb muscles pre- and post-exercise (n=5) and e, hind-limb muscles post-exercise of mdx treated with PDE5A inhibitor before exercise (n=5). White arrowheads mark areas of increased water compartmentalization. f, Percent muscle edema area preand post-exercise and +/− PDE5A inhibitor treatment in mdx mice compared to that of wild-type. (wild-type and mdx n=3, mdx + PDE5A inhibitor n=5, error bars are s.e.m.)

Figure 4. nNOS is reduced in human muscle diseases

Representative immunofluorescent staining in various human muscle diseases: in primary dystrophinopathies, Duchenne and Becker muscular dystrophy (DMD and BMD, respectively); in several forms of limb-girdle muscular dystrophy (LGMD); in two congenital muscular dystrophies (CMD) caused by mutations in extracellular matrix proteins [Ullrich CMD (UCMD), collagen VI and merosin-deficient CMD (MDC1A), laminin-2]. Asterisks mark the same muscle fibers in some of the adjacent panels. (Scale bar = 100 μm)