Tadalafil alleviates muscle ischemia in patients with Becker muscular dystrophy

Abstract

Becker muscular dystrophy (BMD) is a progressive X-linked muscle wasting disease for which there is no treatment. Like Duchenne muscular dystrophy (DMD), BMD is caused by mutations in the gene encoding dystrophin, a structural cytoskeletal protein that also targets other proteins to the muscle sarcolemma. Among these is neuronal nitric oxide synthase (nNOSμ), which requires certain spectrin-like repeats in dystrophin's rod domain and the adaptor protein α-syntrophin to be targeted to the sarcolemma. When healthy skeletal muscle is subjected to exercise, sarcolemmal nNOSμ-derived NO attenuates local α-adrenergic vasoconstriction, thereby optimizing perfusion of muscle. We found previously that this protective mechanism is defective-causing functional muscle ischemia-in dystrophin-deficient muscles of the mdx mouse (a model of DMD) and of children with DMD, in whom nNOSμ is mislocalized to the cytosol instead of the sarcolemma. We report that this protective mechanism also is defective in men with BMD in whom the most common dystrophin mutations disrupt sarcolemmal targeting of nNOSμ. In these men, the vasoconstrictor response, measured as a decrease in muscle oxygenation, to reflex sympathetic activation is not appropriately attenuated during exercise of the dystrophic muscles. In a randomized placebo-controlled crossover trial, we show that functional muscle ischemia is alleviated and normal blood flow regulation is fully restored in the muscles of men with BMD by boosting NO-cGMP (guanosine 3',5'-monophosphate) signaling with a single dose of the drug tadalafil, a phosphodiesterase 5A inhibitor. These results further support an essential role for sarcolemmal nNOSμ in the normal modulation of sympathetic vasoconstriction in exercising human skeletal muscle and implicate the NO-cGMP pathway as a putative new target for treating BMD.

Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1

Functional sympatholysis is impaired in patients with Becker muscular dystrophy (BMD). (A) As shown in the representative trace from a healthy control subject, the lower body negative pressure (LBNP)-induced decrease in forearm muscle oxygenation (HbO2 + MbO2) is greatly attenuated during mild handgrip exercise, demonstrating functional sympatholysis (i.e., normal exercise-induced attenuation of reflex vasoconstriction; grey oval). (B) The representative trace from a patient with BMD shows that sympatholysis is impaired, because handgrip fails to attenuate the LBNP response (red oval) indicating functional muscle ischemia. At the end of each experiment, an arm cuff was inflated to suprasystolic pressure to occlude the forearm circulation, producing a maximal decrease in muscle oxygenation to calculate total labile signal (TLS). (C, D) Summary data for 7 healthy controls (C) and 10 patients with BMD (D) expressed as a percentage of TLS (mean ± SEM). *P<0.01 rest vs. exercise using a linear mixed-effects model.

Fig. 2

Tadalafil alleviates functional muscle ischemia in a patient with Becker muscular dystrophy (BMD). (A) Tadalafil restores functional sympatholysis in a patient with BMD, as shown by the exercise-induced attenuation of the decrease in muscle oxygenation (HbO2+MbO2) with lower body negative pressure (LBNP) (grey oval). (B) In contrast, a placebo is without effect in the same patient, as shown by comparable LBNP responses during rest and exercise (red oval). (C, D) Summary data expressed as a percentage of total labile signal (TLS) (mean ± SEM) from 10 patients with BMD all of whom completed the double-blind cross-over trial on tadalafil (C) and then placebo (D) or vice versa. *P<0.01, treatment condition × exercise condition interaction using a linear mixed-effects model.

Fig. 3

Patient-specific data showing the consistency of the tadalafil effect. (A) With tadalafil, functional sympatholysis (exercise-induced attenuation of reflex vasoconstriction) is evident in nine of 10 patients with Becker muscular dystrophy (BMD), because the lower body negative pressure (LBNP)-induced decrease in forearm muscle oxygenation (HbO2+MbO2) is less during exercise (grey bars) than at rest (black bars). The exception was patient 10 (P10) in whom an adequate tadalafil blood concentration was achieved. (B) With placebo, the LBNP response is virtually identical during rest and exercise for each patient, except patient P9 in whom sympatholysis is clearly evident without tadalafil and possibly in patient P2 in whom sympatholysis is greatly augmented by tadalafil. Data are expressed as a percentage of total labile signal (TLS) (mean ± SEM).

Fig. 4

Immunohistochemistry of muscle biopsies from BMD patients. Muscle biopsy sections stained with haematoxylin and eosin (upper panel) from patient P5 (deletion of exons 45–48) and patient P9 (deletion of exons 14–44) show typical dystrophic hallmarks including variation in muscle fiber size, internal nuclei, fibrosis, and increase in fat tissue. Staining for dystrophin C-terminus (DYS-C) is present in muscle from both P5 and P9. Cytoplasmic staining for nNOS is evident in both P5 and P9 but not in control muscle. Sarcolemmal nNOS labeling is present in P9 but not detected in P5. Bottom panel shows higher magnification of fields enclosed in rectangles. Scale bars are 100 μm.