Kendra Anderson

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

Signaling via the neuronal NOS (nNOS) splice variant nNOSmu is essential for skeletal muscle health and is commonly reduced in neuromuscular disease. nNOSmu 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 nNOSbeta, localized at the Golgi complex in mouse skeletal muscle cells. In contrast to muscles lacking nNOSmu alone, muscles missing both nNOSmu and nNOSbeta were severely myopathic, exhibiting structural defects in the microtubule cytoskeleton, Golgi complex, and mitochondria. Skeletal muscles lacking both nNOSmu and nNOSbeta were smaller in mass, intrinsically weak, highly susceptible to fatigue, and exhibited marked postexercise weakness. Our data indicate that nNOSbeta 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 nNOSmu to the sarcolemma and nNOSbeta to the Golgi. We have previously shown that sarcolemmal nNOSmu matches the blood supply to the metabolic demands of active muscle. We now demonstrate that nNOSbeta 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.

Functional Deficits in nNOSμ-Deficient Skeletal Muscle: Myopathy in nNOS Knockout Mice

Skeletal muscle nNOSμ (neuronal nitric oxide synthase mu) localizes to the sarcolemma through interaction with the dystrophin-associated glycoprotein (DAG) complex, where it synthesizes nitric oxide (NO). Disruption of the DAG complex occurs in dystrophinopathies and sarcoglycanopathies, two genetically distinct classes of muscular dystrophy characterized by progressive loss of muscle mass, muscle weakness and increased fatigability. DAG complex instability leads to mislocalization and downregulation of nNOSμ; but this is thought to play a minor role in disease pathogenesis. This view persists without knowledge of the role of nNOS in skeletal muscle contractile function in vivo and has influenced gene therapy approaches to dystrophinopathy, the majority of which do not restore sarcolemmal nNOSμ. We address this knowledge gap by evaluating skeletal muscle function in nNOS knockout (KN1) mice using an in situ approach, in which the muscle is maintained in its normal physiological environment. nNOS-deficiency caused reductions in skeletal muscle bulk and maximum tetanic force production in male mice only. Furthermore, nNOS-deficient muscles from both male and female mice exhibited increased susceptibility to contraction-induced fatigue. These data suggest that aberrant nNOSμ signaling can negatively impact three important clinical features of dystrophinopathies and sarcoglycanopathies: maintenance of muscle bulk, force generation and fatigability. Our study suggests that restoration of sarcolemmal nNOSμ expression in dystrophic muscles may be more important than previously appreciated and that it should be a feature of any fully effective gene therapy-based intervention.

The α-Syntrophin PH and PDZ Domains Scaffold Acetylcholine Receptors, Utrophin, and Neuronal Nitric Oxide Synthase at the Neuromuscular Junction

At the neuromuscular junction (NMJ), the dystrophin protein complex provides a scaffold that functions to stabilize acetylcholine receptor (AChR) clusters. Syntrophin, a key component of that scaffold, is a multidomain adapter protein that links a variety of signaling proteins and ion channels to the dystrophin protein complex. Without syntrophin, utrophin and neuronal nitric oxide synthase μ (nNOSμ) fail to localize to the NMJ and the AChRs are distributed abnormally. Here we investigate the contribution of syntrophin domains to AChR distribution and to localization of utrophin and nNOSμ at the NMJ. Transgenic mice expressing α-syntrophin lacking portions of the first pleckstrin homology (PH) domain (ΔPH1a or ΔPH1b) or the entire PDZ domain (ΔPDZ) were bred onto the α-syntrophin null background. As expected the ΔPDZ transgene did not restore the NMJ localization of nNOS. The ΔPH1a transgene did restore postsynaptic nNOS but surprisingly did not restore sarcolemmal nNOS (although sarcolemmal aquaporin-4 was restored). Mice lacking the α-syntrophin PDZ domain or either half of the PH1 domain were able to restore utrophin to the NMJ but did not correct the aberrant AChR distribution of the α-syntrophin knock-out mice. However, mice expressing both the transgenic ΔPDZ and the transgenic ΔPH1a constructs did restore normal AChR distribution, demonstrating that both domains are required but need not be confined within the same protein to function. We conclude that the PH1 and PDZ domains of α-syntrophin work in concert to facilitate the localization of AChRs and nNOS at the NMJ.

Loss of nNOS inhibits compensatory muscle hypertrophy and exacerbates inflammation and eccentric contraction-induced damage in mdx mice

Approaches targeting nitric oxide (NO) signaling show promise as therapies for Duchenne and Becker muscular dystrophies. However, the mechanisms by which NO benefits dystrophin-deficient muscle remain unclear, but may involve nNOSβ, a newly discovered enzymatic source of NO in skeletal muscle. Here we investigate the impact of dystrophin deficiency on nNOSβ and use mdx mice engineered to lack nNOSμ and nNOSβ to discern how the loss of nNOS impacts dystrophic skeletal muscle pathology. In mdx muscle, nNOSβ was mislocalized and its association with the Golgi complex was reduced. nNOS depletion from mdx mice prevented compensatory skeletal muscle cell hypertrophy, decreased myofiber central nucleation and increased focal macrophage cell infiltration, indicating exacerbated dystrophic muscle damage. Reductions in muscle integrity in nNOS-null mdx mice were accompanied by decreases in specific force and increased susceptibility to eccentric contraction-induced muscle damage compared with mdx controls. Unexpectedly, muscle fatigue was unaffected by nNOS depletion, revealing a novel latent compensatory mechanism for the loss of nNOS in mdx mice. Together with previous studies, these data suggest that localization of both nNOSμ and nNOSβ is disrupted by dystrophin deficiency. They also indicate that nNOS has a more complex role as a modifier of dystrophic pathology and broader therapeutic potential than previously recognized. Importantly, these findings also suggest nNOSβ as a new drug target and provide a new conceptual framework for understanding nNOS signaling and the benefits of NO therapies in dystrophinopathies.