Re-REVISED paper:E-90280-2008R2 Contractile C2C12 Myotube Model for Studying Exercise-inducible Responses in Skeletal Muscle

Adequate exercise leads to a vast variety of physiological changes in skeletal muscle as well as other tissues/organs and is also responsible for maintaining healthy muscle displaying enhanced insulin-responsive glucose uptake via GLUT4 translocation. We generated highly developed contractile C2C12 myotubes, by manipulating intracellular Ca 2+ transients with electric pulse stimulation (EPS), that are endowed with properties similar to those of in vivo skeletal muscle in terms of (1) excitation-induced contractile activity as a result of de novo sarcomere formation, (2) activation of both the AMP-kinase and stress-activated MAP-kinase cascades and (3) improved insulin responsiveness as assessed by GLUT4 recycling. Tbc1d1, a Rab-GAP implicated in exercise-induced GLUT4 translocation in skeletal muscle, also appeared to be phosphorylated on Ser231 after EPS-induced contraction. In addition, a switch in myosin heavy chain (MHC) expression from “fast-type” to “slow-type” was observed in the C2C12 myotubes endowed with EPS-induced repetitive contractility. Taking advantage of these highly developed contractile C2C12 myotubes, we identified myotube-derived factors responsive to EPS-evoked contraction including the CXC chemokines CXCL1/KC and CXCL5/LIX, as well as IL-6, previously reported to be up-regulated in contracting muscles in vivo. Importantly, animal treadmill experiments revealed that exercise significantly increased systemic levels of CXCL1/KC, perhaps derived from contracting muscle. Taken together, these results confirm that we have established a specialized muscle cell culture model allowing contraction-inducible cellular responses to be explored. Utilizing this model, we identified contraction-inducible myokines potentially linked to the metabolic alterations, immune responses and angiogenesis induced by exercise.