Effect of denervation on titin in myofibrils of rat skeletal muscles

Abstract

Denervated muscles undergo weight loss, cell death, and ATPase activity reduction. These factors cause a fall in tetanic force output and an increase in twitch duration in atrophic muscles. Although an increase in twitch half-relaxation time is also observed, the relaxed processes of myofibrils are still a mystery. Recently, the restoration of force of myocytes after contraction is found to originate from titin, a giant myofibril protein. It is important to determine whether titin also contributes to the restoration of force in skeletal muscle and whether the quantity of titin declines after denervation. Ultrastructural studies show that Z-lines appear degraded in atrophying muscle. Titin interact with &-actinin, the major protein in Z-line region. The major objective of this study was to quantify the changes of titin and &-actinin in myofibrils in rat soleus and anterior tibialis muscles denervated by segmental resection of the sciatic nerve. Using SDS-PAGE to separate myofibril proteins and densitometry to analyze the amounts of titin, myosin, and actin in myofibrils, we found that the titin/myosin ratio decreased in soleus and anterior tibialis muscles after four-weeks of denervation. Immunofluorescence and immunoelectron microscopy for titin with monoclonal antibody T12 which specifically labels a titin epitope near the Z-line displayed both the striated staining pattern observed in normal myofibrils, and also a spotty staining pattern near the Z-line region in denervated myofibrils. These results suggested that titin declined faster than myosin and actin after denervation. This finding can be correlated with sarcomere disassembly, the force output decrease and relaxtion time elongation observed in denervated muscles. Detailed studies on atrophic processes of denervated muscle may provide insights on treatment of muscular dystrophy.