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Autophagy activation in COL6 myopathic patients by a low-protein-diet pilot trial.

Autophagy (2016-09-23)
Silvia Castagnaro, Camilla Pellegrini, Massimo Pellegrini, Martina Chrisam, Patrizia Sabatelli, Silvia Toni, Paolo Grumati, Claudio Ripamonti, Loredana Pratelli, Nadir M Maraldi, Daniela Cocchi, Valeria Righi, Cesare Faldini, Marco Sandri, Paolo Bonaldo, Luciano Merlini
ABSTRACT

A pilot clinical trial based on nutritional modulation was designed to assess the efficacy of a one-year low-protein diet in activating autophagy in skeletal muscle of patients affected by COL6/collagen VI-related myopathies. Ullrich congenital muscular dystrophy and Bethlem myopathy are rare inherited muscle disorders caused by mutations of COL6 genes and for which no cure is yet available. Studies in col6 null mice revealed that myofiber degeneration involves autophagy defects and that forced activation of autophagy results in the amelioration of muscle pathology. Seven adult patients affected by COL6 myopathies underwent a controlled low-protein diet for 12 mo and we evaluated the presence of autophagosomes and the mRNA and protein levels for BECN1/Beclin 1 and MAP1LC3B/LC3B in muscle biopsies and blood leukocytes. Safety measures were assessed, including muscle strength, motor and respiratory function, and metabolic parameters. After one y of low-protein diet, autophagic markers were increased in skeletal muscle and blood leukocytes of patients. The treatment was safe as shown by preservation of lean:fat percentage of body composition, muscle strength and function. Moreover, the decreased incidence of myofiber apoptosis indicated benefits in muscle homeostasis, and the metabolic changes pointed at improved mitochondrial function. These data provide evidence that a low-protein diet is able to activate autophagy and is safe and tolerable in patients with COL6 myopathies, pointing at autophagy activation as a potential target for therapeutic applications. In addition, our findings indicate that blood leukocytes are a promising noninvasive tool for monitoring autophagy activation in patients.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Triton X-100, laboratory grade
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Triton X-100, BioXtra
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IGEPAL® CA-630, for molecular biology
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Phosphatase Inhibitor Cocktail 2, aqueous solution (dark coloration may develop upon storage, which does not affect the activity)
Roche
In Situ Cell Death Detection Kit, TMR red, sufficient for ≤50 tests
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Anti-LC3B antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
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Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5, clone 6C5, Chemicon®, from mouse