Dystrophin R16/17 protein therapy restores sarcolemmal nNOS in trans and improves muscle perfusion and function.

BACKGROUND: Delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma leads to functional muscle ischemia. This contributes to the pathogenesis in cachexia, aging and muscular dystrophy. Mutations in the gene encoding dystrophin result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). In many BMD patients and DMD patients that have been converted to BMD by gene therapy, sarcolemmal nNOS is missing due to the lack of dystrophin nNOS-binding domain. METHODS: Dystrophin spectrin-like repeats 16 and 17 (R16/17) is the sarcolemmal nNOS localization domain. Here we explored whether R16/17 protein therapy can restore nNOS to the sarcolemma and prevent functional ischemia in transgenic mice which expressed an R16/17-deleted human micro-dystrophin gene in the dystrophic muscle. The palmitoylated R16/17.GFP fusion protein was conjugated to various cell-penetrating peptides and produced in the baculovirus-insect cell system. The best fusion protein was delivered to the transgenic mice and functional muscle ischemia was quantified. RESULTS: Among five candidate cell-penetrating peptides, the mutant HIV trans-acting activator of transcription (TAT) protein transduction domain (mTAT) was the best in transferring the R16/17.GFP protein to the muscle. Systemic delivery of the mTAT.R16/17.GFP protein to micro-dystrophin transgenic mice successfully restored sarcolemmal nNOS without inducing T cell infiltration. More importantly, R16/17 protein therapy effectively prevented treadmill challenge-induced force loss and improved muscle perfusion during contraction. CONCLUSIONS: Our results suggest that R16/17 protein delivery is a highly promising therapy for muscle diseases involving sarcolemmal nNOS delocalizaton.

Exercise training and peripheral arterial disease.

Peripheral arterial disease (PAD) is a common vascular disease that reduces blood flow capacity to the legs of patients. PAD leads to exercise intolerance that can progress in severity to greatly limit mobility, and in advanced cases leads to frank ischemia with pain at rest. It is estimated that 12 to 15 million people in the United States are diagnosed with PAD, with a much larger population that is undiagnosed. The presence of PAD predicts a 50% to 1500% increase in morbidity and mortality, depending on severity. Treatment of patients with PAD is limited to modification of cardiovascular disease risk factors, pharmacological intervention, surgery, and exercise therapy. Extended exercise programs that involve walking approximately five times per week, at a significant intensity that requires frequent rest periods, are most significant. Preclinical studies and virtually all clinical trials demonstrate the benefits of exercise therapy, including improved walking tolerance, modified inflammatory/hemostatic markers, enhanced vasoresponsiveness, adaptations within the limb (angiogenesis, arteriogenesis, and mitochondrial synthesis) that enhance oxygen delivery and metabolic responses, potentially delayed progression of the disease, enhanced quality of life indices, and extended longevity. A synthesis is provided as to how these adaptations can develop in the context of our current state of knowledge and events known to be orchestrated by exercise. The benefits are so compelling that exercise prescription should be an essential option presented to patients with PAD in the absence of contraindications. Obviously, selecting for a lifestyle pattern that includes enhanced physical activity prior to the advance of PAD limitations is the most desirable and beneficial.