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NO more muscle fatigue
Ahlke Heydemann and Elizabeth McNally
Section of Cardiology, Department of Medicine, University of Chicago, Chicago, Illinois, USA.
Address correspondence to: Elizabeth McNally, Department of Medicine, Section of Cardiology, The University of Chicago, 5841 S. Maryland, MC6088, Chicago, Illinois 60637, USA. Phone: (773) 702-2672; Fax: (773) 702-2681; E-mail: emcnally@uchicago.edu.
Find articles by Heydemann, A. in: JCI | PubMed | Google Scholar
Section of Cardiology, Department of Medicine, University of Chicago, Chicago, Illinois, USA.
Address correspondence to: Elizabeth McNally, Department of Medicine, Section of Cardiology, The University of Chicago, 5841 S. Maryland, MC6088, Chicago, Illinois 60637, USA. Phone: (773) 702-2672; Fax: (773) 702-2681; E-mail: emcnally@uchicago.edu.
Find articles by McNally, E. in: JCI | PubMed | Google Scholar
Published February 23, 2009 - More info
J Clin Invest. 2009;119(3):448–450. https://doi.org/10.1172/JCI38618.
© 2009 The American Society for Clinical Investigation
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Abstract
Sarcolemma-associated neuronal NOS (nNOS) plays a critical role in normal muscle physiology. In Duchenne muscular dystrophy (DMD), the loss of sarcolemmal nNOS leads to functional ischemia and muscle damage; however, the mechanism of nNOS subcellular localization remains incompletely understood. According to the prevailing model, nNOS is recruited to the sarcolemma by syntrophin, and in DMD this localization is altered. Intriguingly, the presence of syntrophin on the membrane does not always restore sarcolemmal nNOS. Thus, we wished to determine whether dystrophin functions in subcellular localization of nNOS and which regions may be necessary. Using in vivo transfection of dystrophin deletion constructs, we show that sarcolemmal targeting of nNOS was dependent on the spectrin-like repeats 16 and 17 (R16/17) within the rod domain. Treatment of mdx mice (a DMD model) with R16/17-containing synthetic dystrophin genes effectively ameliorated histological muscle pathology and improved muscle strength as well as exercise performance. Furthermore, sarcolemma-targeted nNOS attenuated α-adrenergic vasoconstriction in contracting muscle and improved muscle perfusion during exercise as measured by Doppler and microsphere circulation. In summary, we have identified the dystrophin spectrin-like repeats 16 and 17 as a novel scaffold for nNOS sarcolemmal targeting. These data suggest that muscular dystrophy gene therapies based on R16/17-containing dystrophins may yield better clinical outcomes than the current therapies.
Authors
Yi Lai, Gail D. Thomas, Yongping Yue, Hsiao T. Yang, Dejia Li, Chun Long, Luke Judge, Brian Bostick, Jeffrey S. Chamberlain, Ronald L. Terjung, Dongsheng Duan
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Dystrophin localizes neuronal NOS to the plasma membrane of muscle
The dystrophin-associated protein complex (Figure 1) is found at the plasma membrane of skeletal muscle, where it provides stability to the myofiber membrane during contraction (1). Genetic mutations that ablate dystrophin expression lead to Duchenne muscular dystrophy (DMD) in humans and muscular dystrophy in mdx mice. Dystrophin is a long cytoskeletal protein that contains an actin-binding site at its amino terminus, 23 spectrin-like repeats interrupted by four hinge points, and a carboxyl terminus that links dystrophin to dystroglycan and the other transmembrane and membrane-associated components of the larger protein complex. The syntrophins are cytosolic proteins. α-Syntrophin directly binds to neuronal NOS (nNOS or NOS1) by way of its PDZ (postsynaptic density 95, discs large, and zonula occludens–1) domains (2). Syntrophins bind directly to dystrobrevins and dystrophin, and this interaction was thought to be sufficient to localize nNOS to the plasma membrane (3). In the absence of dystrophin, nNOS is lost from the plasma membrane (2).
Figure 1Mechanism for the localization of nNOS to the plasma membrane of muscle. Sarcolemmal nNOS promotes vasodilation and improves blood flow into muscle. This blood flow is necessary to prevent early fatigue with exercise. In this issue of the JCI, Lai et al. show that a direct interaction between dystrophin and nNOS is mediated by spectrin-like repeats 16 and 17 in dystrophin (SR16/17) (14). It was previously shown that α-syntrophin could directly bind nNOS by way of its PDZ domain (15). Loss of dystrophin in humans with DMD or in mdx mice is associated with reduction in levels of dystrophin-associated proteins including α-syntrophin and nNOS (2). Work by Lai et al. (14) as well as recent reports by Kobayashi et al. (16) and Percival et al. (8) now clarify that loss of nNOS from the plasma membrane mediates muscle fatigue after minimal exercise and that this feature is present in diverse forms of muscle disease.
The functional significance of nNOS displacement from the sarcolemma of muscle was previously shown to be associated with muscle ischemia in both mdx mice and boys with DMD (4, 5). However, mice engineered to lack nNOS itself lack the overt features of muscular dystrophy, such as muscle degeneration, reactive regeneration, and fibrofatty replacement of muscle (6, 7). In earlier studies, the phenotype of mice lacking both nNOS and dystrophin was no different from that observed in the mdx model itself (6, 7).
However, recent reevaluation of nNOS-null mice shows that male nNOS-null mice have smaller muscle mass and reduced force production compared with strain- and sex-matched WT mice (8). When normalized for the smaller muscle mass, force production in nNOS-null mice is normal. However, nNOS-null mice display a specific deficit in adapting to exercise and develop profound fatigue upon repeated muscle contraction. Thus, loss of nNOS or mislocalization of nNOS from its normal position at the plasma membrane causes fatigue with exercise. Interestingly, muscle expressing syntrophin that lacked an nNOS-binding site was also found to exhibit ischemia with exercise (9).
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Minidystrophins only partially correct muscular dystrophy
DMD develops when dystrophin is absent, and gene replacement strategies have been pursued through both viral delivery and transgenesis. Internal truncations of dystrophin that leave the amino and carboxyl termini intact occur naturally with the milder muscle disease Becker muscular dystrophy (BMD). BMD patients display less muscle degeneration than DMD patients but frequently note exercise-induced fatigue (10). Gene replacement therapy for DMD is limited by vector capacity, as the dystrophin protein is large in size. Therefore, smaller dystrophins called mini- or microdystrophins have been engineered to mimic what is naturally found in BMD patients and have been extensively tested in the mdx mouse (11, 12). Gene replacement studies have shown that dystrophin lacking the middle portion of the protein can correct underlying pathology as long as the actin-binding domain and carboxyterminal domains are intact (13).
As now shown by Lai and colleagues in this issue of the JCI, this strategy, while successful at correcting histopathology, leaves the treated animals unable to exercise to the same extent as normal mice (14). To explain the exercise deficit, they noted that nNOS was not normally positioned at the plasma membrane in mdx mice expressing minidystrophins. Specifically, the authors correlated the absence of dystrophin’s spectrin-like repeats 16 and 17 with absent plasma membrane nNOS and an inability to undergo exercise conditioning. Moreover, these mice, referred to as ΔH2–R19 transgenic mdx mice, develop skeletal muscle ischemia with exercise. Ischemia and the absence of spectrin-like repeats 16 and 17 correlated with the inability to improve exercise capacity over time (summarized in Table 1). They concluded that association of nNOS with the dystrophin complex requires spectrin-like repeats 16 and 17 and is necessary to produce vasodilation and supply oxygen to exercising muscle. It was previously shown that the syntrophin PDZ domain was sufficient to anchor nNOS (15). Together, these findings indicate that nNOS requires both dystrophin and syntrophin for full localization and function at the plasma membrane.
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