NO skeletal muscle derived relaxing factor in Duchenne muscular dystrophy

The remarkable discovery of an endothelial-derived relaxing factor by Robert Furchgott in 1980 initiated studies that ultimately led to identification of nitric oxide (NO) as an endogenous signaling molecule (1–4). In the classical pathway described by Furchgott, NO is synthesized by vascular endothelial cells and then diffuses into the underlying smooth muscle cells to mediate vessel relaxation. Although this particular pathway plays a major role in vascular control, subsequent work has shown that endothelium-dependent relaxation is not the only mode for NO to regulate blood flow. In certain vascular beds, regulation of vessel tone by NO does not require the endothelium but instead relies on neuronal activity. Indeed, molecular cloning of NO synthase (NOS) established that not only endothelial cells but also neurons synthesize constitutively (5). For example, high density of NOS in the nerve plexus of the corpora cavernosum regulates penile blood flow, and NOS activity is essential for erection (6). In fact, sildenafil (Viagra) enhances erectile function by specifically augmenting vasodilatory actions of neuron-derived NO (7).

Dynamic regulation of blood flow by NO is not the prerogative of endothelial cells and neurons. The paper by Thomas et al. in this issue of the Proceedings (8) demonstrates that contracting muscle cells also produce vasoactive NO. These new findings help explain how skeletal muscle activity increases blood flow to support the heavy metabolic load. Though contracting skeletal muscle has long been known to produce a variety of vasoactive substances, the identity and roles of these factors have remained uncertain (9, 10). The work by Thomas et al. demonstrates that NO, produced by nNOS activity in contracting skeletal muscle, opposes an otherwise deleterious adrenergic vasoconstriction (6). As this protective NOS pathway is disrupted in Duchenne muscular dystrophy, the present work has important implications for both pathophysiology and possible treatment of muscle disease.

Neuronal NOS in Skeletal Muscle

Molecular studies originally demonstrated that NO in skeletal muscle derives from the neuronal NOS isoform (nNOS) (11), which is alternatively spliced in skeletal muscle to yield nNOSμ (12). Immunohistochemical analysis indicates that nNOSμ is specifically enriched in fast-twitch muscle fibers (13), which release NO is response to muscle contraction (14). This muscle-derived NO mediates several physiological actions on skeletal muscle. NO profoundly increases glucose uptake into muscle cells (15). This effect of NO appears to be specific for exercise-induced glucose uptake, as NO has little effect on insulin-mediated glucose transport (16).

NO produced in active skeletal muscle also modulates contractile force (13). Similar to its actions in smooth muscle, NO appears to inhibit contractility force in skeletal muscle. Thus, isometric forces produced during twitch and submaximal tetanic contractions are increased by NOS inhibitors and are depressed by NO donors (13). Relaxation of smooth muscle by NO is mediated by increases in cGMP, and a similar mechanism operates in skeletal muscle. However, in skeletal muscle, the magnitude of cGMP-dependent effects is modest, suggesting that NO also can influence contractile force directly. One molecular target for direct actions of NO is the sarcoplasmic reticulum calcium release channel, which is highly redox sensitive and can be gated by NO (17).

NO Regulates Skeletal Muscle Blood Flow

In addition to mediating autocrine actions on skeletal muscle metabolism and contractile force, NO appears to regulate blood flow to skeletal muscle (18). During exercise, blood flow rapidly increases in contracting muscles to accommodate the elevated metabolic demands of the tissue (10). This exercise-induced hyperemia is blunted by NOS inhibitors, suggesting a role for NO in this response (19–21). Because NOS inhibitors do not completely block exercise hyperemia, NO must play a modulatory rather than an absolute role in this response (18).

One critical component of the vascular response to exercise is the attenuation of sympathetic vasoconstriction that occurs in contracting muscle. Because muscle contraction reflexly increases sympathetic drive to skeletal muscle, exercise hyperemia requires that vessels in contracting muscles are less responsive to the sympathetic discharge. Experiments in intact rodent models indicated that this exercise-induced sympatholysis occurs predominately in fast-twitch muscle groups and appears to be mediated by a local metabolite (22, 23). Pharmacological studies with NOS inhibitors have shown that NO is responsible for exercise-induced sympatholysis, but the cellular source of the NO has remained unclear (24). This uncertainty is caused by the fact that NOS inhibitors do not discriminate between constitutive NOS isoforms, so that the NO could derive either from endothelial NOS (eNOS) in the vasculature or from nNOS in the skeletal myofibers. To identify the source of vasoactive NO in this pathway, Thomas et al. have evaluated skeletal muscle blood flow responses in mutant mice lacking nNOS (8). The authors found that exercise-induced hyperemia generally is preserved in the nNOS knockouts, but the nNOS knockouts specifically lack contraction-induced suppression of adrenergic vasoconstriction. These experiments definitively demonstrate that skeletal muscle-derived NO mediates sympatholysis. Also, because nNOS is enriched in fast-twitch muscle fibers (13), these results explain why exercise-induced sympatholysis occurs predominately in fast-twitch fibers.

NO and Muscular Dystrophy

A striking aspect of nNOS in skeletal muscle is the cellular localization of the synthase at the plasma membrane (13). Targeting the synthase to the plasma membrane serves at least two purposes. First, this localization allows depolarization and subsequent calcium influx across the plasma membrane to regulate nNOS activity through calcium and calmodulin. Second, synthesis of NO at the muscle plasma membrane facilitates delivery of NO to surrounding cellular targets such as the vasculature.

nNOS associates with the plasma membrane by binding a protein complex containing dystrophin, the gene mutated in Duchenne muscular dystrophy (25). nNOS is selectively lost from the plasma membrane of muscle from patients with Duchenne dystrophy and from mdx mice, which lack dystrophin. nNOS associates with syntropin, a cytosolic dystrophin- binding protein, rather than dystrophin itself (26). Interaction of nNOS with synthrophin is mediated by PDZ protein motifs near the N termini of nNOS and syntrophin.

The absence of nNOS may contribute to the pathology in Duchenne dystrophy (25, 27). As evidence for this, nNOS is the only component of the dystrophin complex that is specifically enriched in fast-twitch muscle cells, and these cells preferentially degenerate in Duchenne dystrophy (28). However, it is uncertain how the loss of skeletal muscle in nNOS might contribute to pathophysiology. To address this, Thomas et al. (8) evaluate skeletal muscle blood flow responses in a mouse model of Duchenne dystrophy, the mdx mouse. Strikingly, contraction fails to suppress adrenergic vasoconstriction in mdx mice, just like nNOS knockout mice. Additionally, the contractin-induced increase in cGMP and inhibition of smooth muscle myosin light chain phosphorylation are compromised in fast-twitch muscles from mdx mice (29). Aside from lacking this exercise-induced sympatholytic response, blood flow in the mdx mice is normal, which is similar to what is observed in nNOS knockouts.

Taken together, these studies demonstrate that NO plays a major role in regulating blood flow to exercising muscle by blunting the constrictive response to an otherwise adverse increase in sympathetic tone. Through this mechanism, NO may help muscle to tolerate heavy exercise. In this regard, it is interesting to note that nNOS protein levels in muscle dramatically increase after exercise (16, 30).

This work also suggests that an abnormal vascular response may mediate pathophysiological aspects of Duchenne muscular dystrophy. Although nNOS knockout mice do not themselves show pathological changes characteristic of muscular dystrophy (31, 32), this may reflect a limitation of the mouse model. In humans with Becker muscular dystrophy, which is the result of specific mutations of dystrophin, the absence of nNOS, but not other components of the dystrophin complex, correlates closely with the severity of the disease (33). The absence of nNOS in Duchenne and Becker muscular dystrophies subjects the contracting muscle to hypoperfusion and ischemia caused by unopposed adrenergic vasoconstriction. As fast-twitch muscle fibers highly depend on nutrient delivery and are not exercise tolerant, this mechanism may contribute to explaining the preferential destruction of these fibers in the initial stages of Duchenne muscular dystrophy (28). Manipulating NO levels in muscle therefore may represent a possible strategy for treatment of muscular dystrophy.