Analysis of myotilin turnover provides mechanistic insight into the role of myotilinopathy-causing mutations.

MFM (myofibrillar myopathies) are caused by mutations in several sarcomeric components, including the Z-disc protein myotilin. The morphological changes typical of MFM include Z-disc alterations and aggregation of dense filamentous sarcomeric material. The causes and mechanisms of protein aggregation in myotilinopathies and other forms of MFM remain unknown, although impaired degradation may explain, in part, the abnormal protein accumulation. In the present paper we have studied the mechanisms regulating myotilin turnover, analysed the consequences of defective myotilin degradation and tested whether disease-causing myotilin mutations result in altered protein turnover. The results indicate that myotilin is a substrate for the Ca(2+)-dependent protease calpain and identify two calpain cleavage sites in myotilin by MS. We further show that myotilin is degraded by the proteasome system in transfected COS7 cells and in myotubes, and that disease-causing myotilinopathy mutations result in reduced degradation. Finally, we show that proteolysis-inhibitor-induced reduction in myotilin turnover results in formation of intracellular myotilin and actin-containing aggregates, which resemble those seen in diseased muscle cells. These findings identify for the first time biological differences between wt (wild-type) and mutant myotilin. The present study provides novel information on the pathways controlling myotilin turnover and on the molecular defects associated with MFM.