Defective Autophagic Degradation in Aged D2‐mdx Diaphragms

Abstract

Duchenne muscular dystrophy (DMD) is a progressive, fatal, muscle degenerative disease caused by the absence of dystrophin, which follows an X‐linked recessive inheritance pattern. Among other cellular dysfunctions, impaired autophagy has been identified as a consequence of dystrophin deficiency in humans. Accordingly, we hypothesized that degradation of autophagosomes would be impaired in muscles of D2‐mdx mice, an emerging DMD model. To test this hypothesis, diaphragm muscles from 11 month old D2‐mdx (diseased) and DBA (healthy) mice were collected, protein extracted, and western blot analysis was performed. Data were compared with a Student’s t‐test and significance was established as p<0.05. Among the markers of upstream activation of autophagy, total AMPK was increased by 3.5‐fold, phosphorylated (p)‐AMPK was similar, and p‐AMPK/AMPK was decreased by 80% in dystrophic muscle compared to healthy muscle. Similarly, total ULK1 was increased 1.7‐fold in muscle from D2‐mdx compared to DBA, while p‐ULK1 and p‐ULK1/ULK1 were similar between groups. Total Beclin1 was increased 10‐fold and p‐Beclin1 was increased 4‐fold, resulting in a 70% reduction in p‐Beclin1/Beclin1 in dystrophic muscle compared to healthy muscle. Total mTOR, p‐mTOR, and p‐mTOR/mTOR were similar between groups. Autophagosome formation proteins ATG7, ATG16L‐1, ATG12/5 complex, LC3A/B‐I and LC3A/B‐II were increased 2–3‐fold in D2‐mdx compared to DBA, however, the ratio of LC3A/B‐II/I was similar between groups. Relative protein abundance of p62 was increased 2‐fold in D2‐mdx compared to DBA. The relative amounts of the lysosomal membrane proteins LAMP1 and LAMP2 were similar between groups, suggesting similar lysosomal content in diseased and healthy muscles. These data appear to indicate decreased autophagosome degradation in dystrophic muscle compared to healthy. Given the similar lysosome content, autophagic dysfunction in D2‐mdx mice may be due to decreased lysosome fusion or function.Support or Funding InformationSupported in part by Ryan’s Quest, Michael’s cause and Parent Project Muscular Dystrophy (01297)