Muscle specific Ring finger‐1 (MuRF‐1) siRNA in C2C12 cells leads to an increase in Troponin I protein

Abstract

We have previously shown that the E3 ubiquitin ligase, MuRF-1, is elevated in animal models of muscle atrophy, providing evidence for the involvement of MuRF-1 in divergent models of skeletal muscle wasting. MuRF-1 knockout mice show a resistance to denervation-induced atrophy (Bodine et al. 2001), further implicating MuRF-1 activity in the regulation of muscle wasting. Our laboratory is investigating the molecular pathways associated with MuRF-1 action in skeletal muscle. Here we show the effects of MuRF-1 siRNA on Troponin I protein levels in C2C12 cells, a mouse skeletal muscle cell line. C2C12 cells express muscle specific proteins and can be differentiated from myoblasts to myotubes by starving the cells in media with 2% horse serum in place of fetal bovine serum. When C2C12 myoblasts were transfected with MuRF-1 siRNA duplexes (Dharmacon) and allowed to differentiate to myotubes, Troponin I protein levels increased relative to other muscle markers. MuRF-1 overexpression caused a reciprocal decrease in Troponin I levels. These data are consistent with recent reports that cardiac troponin I is a direct ubiquitination target of MuRF-1 (Kedar et al. 2004). Current work is focusing on determining whether direct ubiquitination of Troponin I is related to MuRF-1’s role in muscle catabolism.