Sodium nitrate alleviates functional muscle ischaemia 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. BMD is caused by in-frame mutations in the gene encoding dystrophin, a structural cytoskeletal protein that also targets other proteins to the sarcolemma. Among these is neuronal nitric oxide synthase mu (nNOSμ), which requires specific spectrin-like repeats (SR16/17) in dystrophin's rod domain and the adaptor protein α-syntrophin for sarcolemmal targeting. When healthy skeletal muscle is exercised, sarcolemmal nNOSμ-derived nitric oxide (NO) attenuates α-adrenergic vasoconstriction, thus optimizing perfusion. In the mdx mouse model of dystrophinopathy, this protective mechanism (functional sympatholysis) is defective, resulting in functional muscle ischaemia. Treatment with a NO-donating non-steroidal anti-inflammatory drug (NSAID) alleviates this ischaemia and improves the murine dystrophic phenotype. In the present study, we report that, in 13 men with BMD, sympatholysis is defective mainly in patients whose mutations disrupt sarcolemmal targeting of nNOSμ, with the vasoconstrictor response measured as a decrease in muscle oxygenation (near infrared spectroscopy) to reflex sympathetic activation. Then, in a single-arm, open-label trial in 11 BMD patients and a double-blind, placebo-controlled cross-over trial in six patients, we show that acute treatment with oral sodium nitrate, an inorganic NO donor without a NSIAD moiety, restores sympatholysis and improves post-exercise hyperaemia (Doppler ultrasound). By contrast, sodium nitrate improves neither sympatholysis, nor hyperaemia in healthy controls. Thus, a simple NO donor recapitulates the vasoregulatory actions of sarcolemmal nNOS in BMD patients, and constitutes a putative novel therapy for this disease.

© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Figures

Figure 1. BMD mutations that interrupt sarcolemmal anchoring of nNOS impair functional sympatholysis

Left: representative immunohistochemistry from muscle biopsies from a healthy control subject (A) and two BMD patients (B and C). Muscle biopsy sections stained with haematoxylin and eosin (upper left) from the two BMD patients show typical dystrophic hallmarks, including variation in muscle fibre size, internal nuclei, fibrosis and an increase in fat tissue. Staining for dystrophin C‐terminus (DYS‐C) is present in both BMD patients, although no staining for dystrophin N‐terminus (DYS‐N) is present (evidence for truncated dystrophin). Sarcolemmal nNOS staining is absent in (B) but present in (C). Middle: representative tracing from a healthy control subject (A), a BMD patient lacking sarcolemmal nNOS (B, P2) and a BMD patient with nNOS retained at the sarcolemma (C, P11). A black circle highlights the LBNP‐induced change in forearm muscle oxygenation at rest, whereas a red circle highlights the LBNP‐induced change in forearm muscle oxygenation during exercise. Note that, in healthy controls, the decrease in forearm muscle oxygenation is greatly attenuated (termed functional sympatholysis). At the end of each experiment, an arm cuff was inflated to suprasystolic pressure to occlude the forearm circulation, producing a maximal decrease in forearm muscle oxygenation to calculate the TLS. Right: summary forearm muscle oxygenation data from 13 healthy subjects (A), seven BMD patients in whom sarcolemmal nNOS was absent (B) and six BMD patients in whom sarcolemmal nNOS was present (C). Data are reported as the mean + SE.

Figure 2. Sodium nitrate alleviates functional muscle ischaemia in BMD: acute treatment trial

A, representative tracing (left) from a patient with BMD, showing that sympatholysis is impaired because handgrip fails to attenuate the LBNP‐induced reflex decrease in forearm muscle oxygenation (black circle). At the end of each experiment, an arm cuff was inflated to suprasystolic pressure to occlude the forearm circulation, producing a maximal decrease in forearm muscle oxygenation to calculate the TLS. Right: summary forearm muscle oxygenation data from the 10 patients with BMD studied at rest (black bar) and during exercise (grey bar), expressed as a percentage of TLS. B, representative tracing from the same BMD subject after oral treatment with sodium nitrate, showing that the LBNP‐induced decrease in forearm muscle oxygenation is greatly attenuated during mild handgrip exercise (grey circle). Summary data are shown on the right (n = 10). Of note, we observed no difference between the LBNP response at rest before and after sodium nitrate (not statistically significant). Data are reported as the mean + SE.

Figure 3. Patient‐specific data from the acute treatment trial

A, at baseline, the LBNP‐induced decrease in forearm muscle oxygenation (HbO2 + MbO2) is very consistent at rest (black bar) and during exercise (grey bar) for each patient. B, sodium nitrate restored functional sympatholysis in all but two subjects (P1 and P13). Note that Patient 5 (P5) deviated from the protocol by brushing his teeth during this initial study visit (V1). Accordingly, his data were removed from the analysis. When we repeated the experiment in P11 several months later (V2) (i.e. when the subject was reminded to avoid using toothpaste and mouthwash), the change in plasma nitrite concentration doubled from the first visit, and sympatholysis was improved. Data are expressed as a percentage of the TLS.

Figure 4. Test–retest reproducibility

Four BMD subjects were studied several weeks apart. The data show excellent test–retest reproducibility for functional sympatholysis before sodium nitrate ingestion (showing that functional muscle ischaemia is consistently impaired in four of four patients) and after sodium nitrate ingestion (showing consistent restoration of functional sympatholysis in four of four patients).

Figure 5. Placebo‐controlled cross‐over trial: sodium nitrate alleviates functional muscle ischaemia and increased exercise‐induced hyperaemia in BMD

A, sodium nitrate supplementation restored functional sympatholysis (exercise‐induced attenuation of reflex vasoconstriction, measured by NIR) in men with BMD (n = 6), whereas placebo had no such effect. B, sodium nitrate supplementation does not affect resting skeletal muscle blood flow (measured by brachial artery Doppler ultrasound) but significantly increases exercise‐induced hyperaemia compared to placebo (n = 6). Data are expressed as the mean ± SE.

Figure 6. Sodium nitrate does not improve functional sympatholysis and does not increase exercise‐induced hyperaemia in healthy volunteers

A, sodium nitrate supplementation failed to improve functional sympatholysis (exercise‐induced attenuation of reflex vasoconstriction, measured by NIR) in healthy male volunteers (n = 8). B, sodium nitrate supplementation did not affect resting skeletal muscle blood flow (measured by brachial artery Doppler ultrasound), nor did it affect exercise‐induced hyperaemia (n = 8). Data are expressed as the mean ± SE.

Figure 7. Circulating nitrite serves as an alternative nitric oxide donor only in the presence of deoxyHb and/or deoxyMb

A, rhythmic handgrip exercise significantly increases skeletal muscle deoxyHb and deoxyMb content, as measured by NIR over the flexor digitorum profundus muscle (n = 6). Insert: original NIR record, showing a steep increase in deoxyHb content during the transition from rest to exercise. All data are expressed as a percentage of the TLS. B, sodium nitrate significantly increases circulating plasma nitrite concentration in patients with BMD (n = 6). C, circulating NO2 −is reduced to NO via deoxyHb in the microvascular space, and by deoxyMb in skeletal myocytes, leading to improvements in skeletal muscle blood flow regulation. Data are reported as the mean + SE.