MiRNA expression profiling of skeletal muscles in aged rodents

Skeletal muscle atrophy is one of the critical issues which elderly people face. The precise mechanism underlying muscle atrophy during aging is not fully understood. In order to identify miRNA whose expression is changed in age-associated muscle atrophy, we performed miRNA expression profiling of skeletal muscles in young and aged rats. Microarray analysis revealed differential miRNA expression in EDL and soleus muscles of aged rats compared with those of young rats. We next investigated whether the age-associated changes of miRNA expression observed in rats were recapitulated in mice and found that the expression level of miR-206 in EDL muscle and that of miR-196a in EDL and soleus muscles were respectively higher and lower in aged rodents than in young rodents. In mouse C2C12 myoblasts and myotubes, introduction of miR-196a decreased the protein level of Forkhead-box transcription factor Foxo1, a known target of miR-196a, indicating that miR-196a may regulate Foxo1 expression also in skeletal muscles. Furthermore, miR-196a overexpression exacerbated cell death caused by an exposure to hydrogen peroxide. Lastly, we demonstrated that expression of Foxo1 was elevated in EDL and soleus muscles of aged mice compared with those of young mice. These results suggest that miRNAs are involved in skeletal muscle atrophy during aging and that decreased miR-196a expression may protect skeletal muscle cells from oxidative stress in part through induction of Foxo1. Male Fischer 344 rats were obtained at one month of age from Charles River Laboratories (Yokohama, Japan). Rats were maintained under the standard animal laboratory condition with 12-h light-dark cycle at 23 ºC and were provided water and food ad libitum. Rats were sacrificed at 6-7 and 25-28 months of age and hindlimb muscles were dissected. The weight of the dissected extensor digitorum longus (EDL) muscle, and soleus muscle were measured. Skeletal muscles dissected from rodents were immersed in the RNAlater RNAstabilization reagent (Qiagen, Hilden, Germany) to prevent RNA degradation. The experimental procedures were approved by the Animal Ethics Committee of the Tokyo Metropolitan Institute of Gerontology.