Primary Role of Functional Ischemia, Quantitative Evidence for the Two-Hit Mechanism, and Phosphodiesterase-5 Inhibitor Therapy in Mouse Muscular Dystrophy

Abstract

BackgroundDuchenne Muscular Dystrophy (DMD) is characterized by increased muscle damage and an abnormal blood flow after muscle contraction: the state of functional ischemia. Until now, however, the cause-effect relationship between the pathogenesis of DMD and functional ischemia was unclear. We examined (i) whether functional ischemia is necessary to cause contraction-induced myofiber damage and (ii) whether functional ischemia alone is sufficient to induce the damage.Methodology/Principal Findings In vivo microscopy was used to document assays developed to measure intramuscular red blood cell flux, to quantify the amount of vasodilatory molecules produced from myofibers, and to determine the extent of myofiber damage. Reversal of functional ischemia via pharmacological manipulation prevented contraction-induced myofiber damage in mdx mice, the murine equivalent of DMD. This result indicates that functional ischemia is required for, and thus an essential cause of, muscle damage in mdx mice. Next, to determine whether functional ischemia alone is enough to explain the disease, the extent of ischemia and the amount of myofiber damage were compared both in control and mdx mice. In control mice, functional ischemia alone was found insufficient to cause a similar degree of myofiber damage observed in mdx mice. Additional mechanisms are likely contributing to cause more severe myofiber damage in mdx mice, suggestive of the existence of a “two-hit” mechanism in the pathogenesis of this disease.Conclusions/SignificanceEvidence was provided supporting the essential role of functional ischemia in contraction-induced myofiber damage in mdx mice. Furthermore, the first quantitative evidence for the “two-hit” mechanism in this disease was documented. Significantly, the vasoactive drug tadalafil, a phosphodiesterase 5 inhibitor, administered to mdx mice ameliorated muscle damage.

Drug screening in a zebrafish model of Duchenne muscular dystrophy

“…Two of these inhibitors, sildenafil and tadalafil, have been independently identified by others (20,26), thus validating our screening strategy as a way of finding pathways that might influence the skeletal muscle abnormalities in dystrophin-null fish. The other compounds identified in our screen target additional pathways and represent additional targets for therapy.…”

“…It has been shown that aminophylline has numerous antiinflammatory effects, including the inhibition of inflammatory mediators and activation of NF-κB (22)(23)(24)(25). Previously, other groups have reported that a PDE5 inhibitor restores mdx mouse muscle to normal (20,21,26). It is generally known that PDE inhibitors cause an increase in intracellular cAMP and/or cGMP.…”

nNOS regulation of skeletal muscle fatigue and exercise performance

“…From a therapeutic point of view, vascular dysfunction can be targeted with vasodilating drugs. Pharmacological augmentation of NO-cGMP signaling using phosphodiesterase 5 inhibitors to increase the blood supply to working mdx muscles can reduce post-exercise muscle damage and enhance nNOS-dependent mild exercise-induced inactivity in mdx mice (Asai et al 2007;Kobayashi et al 2008). Together, these data highlight the importance of the vasomodulatory function of sarcolemmal nNOSμ in promoting exercise performance in dystrophic muscle.…”

“…The exercise intolerance shown by DMD patients appears to arise in part from vascular dysfunction, which has long been suspected to be a critical pathogenic mechanism in DMD (Asai et al 2007;Kobayashi et al 2008;Lai et al 2009;reviewed in Percival et al 2011). Sarcolemmal nNOSμ deficiency leads to unopposed α-adrenergic receptor-mediated vasoconstriction in the active hindlimbs of mice mdx and contracting forearms of DMD patients (Thomas et al 1998;Sander et al 2000).…”

Sildenafil and cardiomyocyte-specific cGMP signaling prevent cardiomyopathic changes associated with dystrophin deficiency

“…Sildenafil also significantly reduces exerciseinduced ischemia (33), a potentially important pathogenetic mechanism in dystrophin-deficient skeletal muscle (10,34). In fact, defects in cGMP signaling are also suggested from studies in dystrophin-deficient skeletal muscle (35,36), and recently, Asai et al (37) showed that treatment with tadalafil, another known PDE5 inhibitor, ameliorates contraction-induced skeletal muscle damage. Finally, sildenafil can cross the blood-brain barrier to inhibit brain PDE5A (38), which could theoretically affect sympathetic and parasympathetic outflow.…”