Glial Cell Line Derived Neurotrophic Factor Protein Secretion by Skeletal Muscles is Altered by Activity of L‐Type Calcium Channels

Abstract

Glial cell line‐derived neurotrophic factor (GDNF) is an important signaling molecule throughout the peripheral nervous system. GDNF protein is produced and secreted by skeletal muscle cells and helps maintain motor neuron innervation at the neuromuscular junction. Treatment with exogenous GDNF prevents denervation which occurs with aging and neurodegenerative disease. The therapeutic potential of GDNF cannot be fully explored without understanding the mechanisms by which GDNF protein production is regulated. Although the specific cellular events necessary for regulation of GDNF secretion remains to be elucidated, previous studies from our laboratory have shown that increased skeletal muscle activity significantly alters GDNF protein levels in vivo and in vitro. The primary objective of this study was to determine the role of calcium in regulating GDNF protein secretion by skeletal muscle cells. Skeletal muscle cells (C2C12) were grown in culture and allowed to differentiate into myotubes which display a contractile phenotype. The following treatments were given, Nifedipine to block the voltage‐gated L‐type calcium channels, and Bay K8644 to enhance calcium influx through L‐type calcium channels. Both Western Blot and ELISA were employed for protein identification and GDNF protein quantification respectively. Preliminary results indicate that treatment of myotubes with Bay K8644 (100μM) caused an increase in GDNF secretion to 170% of that in untreated controls. Treatment of cells with Nifedipine blocked the stimulatory effect of Bay K8644, and treatment with Nifedipine alone (10μM and 100μM) inhibited GDNF secretion to 77% of that in control cells. The results suggest that secretion of GDNF protein by C2C12 myotubes is regulated by intracellular calcium levels. Acquiring a greater understanding of the role that calcium plays in regulating GDNF production may help to identify potential sites for therapeutic intervention to increase or decrease GDNF production.Support or Funding InformationNSF Grant DBI‐1062883 and NIH Grant 1R15AG022908‐01A2