Golgi and sarcolemmal neuronal NOS differentially regulate contraction-induced fatigue and vasoconstriction in exercising mouse skeletal muscle
Justin M. Percival, … , Marvin E. Adams, Stanley C. Froehner
Justin M. Percival, … , Marvin E. Adams, Stanley C. Froehner
Published February 1, 2010
Citation Information: J Clin Invest. 2010;120(3):816-826. https://doi.org/10.1172/JCI40736.
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Research Article Muscle biology

Golgi and sarcolemmal neuronal NOS differentially regulate contraction-induced fatigue and vasoconstriction in exercising mouse skeletal muscle

Abstract

Signaling via the neuronal NOS (nNOS) splice variant nNOSμ is essential for skeletal muscle health and is commonly reduced in neuromuscular disease. nNOSμ is thought to be the predominant source of NO in skeletal muscle. Here we demonstrate the existence of what we believe to be a novel signaling pathway, mediated by the nNOS splice variant nNOSβ, localized at the Golgi complex in mouse skeletal muscle cells. In contrast to muscles lacking nNOSμ alone, muscles missing both nNOSμ and nNOSβ were severely myopathic, exhibiting structural defects in the microtubule cytoskeleton, Golgi complex, and mitochondria. Skeletal muscles lacking both nNOSμ and nNOSβ were smaller in mass, intrinsically weak, highly susceptible to fatigue, and exhibited marked postexercise weakness. Our data indicate that nNOSβ is a critical regulator of the structural and functional integrity of skeletal muscle and demonstrate the existence of 2 functionally distinct nNOS microdomains in skeletal muscle, created by the differential targeting of nNOSμ to the sarcolemma and nNOSβ to the Golgi. We have previously shown that sarcolemmal nNOSμ matches the blood supply to the metabolic demands of active muscle. We now demonstrate that nNOSβ simultaneously modulates the ability of skeletal muscle to maintain force production during and after exercise. We conclude therefore that nNOS splice variants are critical regulators of skeletal muscle exercise performance.

Authors

Justin M. Percival, Kendra N.E. Anderson, Paul Huang, Marvin E. Adams, Stanley C. Froehner

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Figure 2

nNOS splice variants and nNOS mutant mouse lines used to analyze NO signaling in skeletal muscle.

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nNOS splice variants and nNOS mutant mouse lines used to analyze NO sign...
(A) Exon structure of the murine Nos1 gene, which encodes 31 exons (black boxes) (39). Relevant isozymes are shown here. The coding sequence is shown in gray, and asterisks mark translation initiation sites. The 5ι- and 3ι-untranslated sequences are shown as white boxes. In skeletal muscle, the predominant transcript is nNOSμ containing exon 2 (encoding the PSD-95, discs-large, ZO-1 [PDZ] domain) and the 34–amino acid μ-insert. nNOSβ is formed by the splicing of exon 1a to exon 3, creating a unique 6–amino acid N terminus. nNOSγ is produced by translation initiation at an internal ATG site in exon 5. (B) WT muscle can express all nNOS splice variants. nNOSμ (yellow) localizes to the sarcolemma by binding α-syntrophin (Syn; green), a member of the dystrophin-glycoprotein complex (gray). nNOSμ also localizes to the cytoplasm. In contracting muscles, sarcolemmal nNOSμ-derived NO overrides sympathetic vasoconstriction maintaining blood vessel dilation (large black arrow) (7). In the present study, we identify a splice variant of nNOS localized to the Golgi complex, presumed to be nNOSβ (red). The small black arrows represent the synthesis of NO from l-arginine by nNOS. (C) Muscles lacking α-syntrophin cannot localize nNOSμ to the sarcolemma and thus cannot attenuate vasoconstriction, despite expression of cytosolic nNOSμ and Golgi nNOSβ. (D) The muscles of KN1 mice do not express nNOSμ, due to deletion of exon 2, but nNOSβ and nNOSγ splice variant production is unaffected (24). (E) KN2 mice do not express any full-length active nNOS splice variants due to deletion of exon 6 encoding the heme-binding domain (25).