Quantitation of autophagic flux in mature skeletal muscle

Reliable and quantitative assays to measure in vivo autophagy are essential. Currently, there are varied methods for monitoring autophagy; however, it is a challenge to measure autophagic flux in an in vivo model system. Conversion and subsequent degradation of the microtubule-associated protein 1 light chain 3 (MAP1-LC3/LC3) to the autophagosome associated LC3-II isoform can be evaluated by immunoblot. However, static levels of endogenous LC3-II protein may render possible misinterpretations since LC3-II levels can increase, decrease or remain unchanged in the setting of autophagic induction. Therefore, it is necessary to measure LC3-II protein levels in the presence and absence of lysomotropic agents that block the degradation of LC3-II, a technique aptly named the autophagometer. In order to measure autophagic flux in mouse skeletal muscle, we treated animals with the microtubule depolarizing agent colchicine. Two days of 0.4 mg/kg/day intraperitoneal colchicine blocked autophagosome maturation to autolysosomes and increased LC3-II protein levels in mouse skeletal muscle by >100%. The addition of an autophagic stimulus such as dietary restriction or rapamycin led to an additional increase in LC3-II above that seen with colchicine alone. Moreover, this increase was not apparent in the absence of a colchicine block. Using this assay, we evaluated the autophagic response in skeletal muscle upon denervation induced atrophy. Our studies highlight the feasibility of performing an in vivo autophagometer study using colchicine in skeletal muscle.