C2c12 muscle cells

To study the effect of circulating factors in mice subjected to LAD ligation, we used C2C12 mouse muscle cells (American Type Culture Collection, Manassas, VA, USA). C2C12 muscle cells were grown in high glucose Dulbeco's Modified Eagle's Medium (DMEM) supplemented with 10% non-heat inactivated fetal bovine serum (FBS, American Type Culture Collection, Manassas, VA, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin in 5% CO2 atmosphere at 37°C. Cells were used at low passage (between passages 2 and 4) for all experiments to maintain the differentiation potential of the cultures. Cells were grown in 10% FBS until they reached approximately 70% confluence, at which time the medium was replaced with DMEM containing 2% FBS for induction of differentiation into myotubes. When myotubes had formed, cytosine arabinoside (10 μM) was added to the culture medium for 48 h in order to remove any remaining dividing myoblasts. Myotubes were treated for 24 h 10% of pooled serum from mice 24h after LAD ligation, in the presence or absence of 1μM of the selective inhibitor of transforming growth factor-β type I receptor GW788388, which inhibits MSTN signaling (Tocris Bioscience, Ellisville, MI, USA) in 0.01% DMSO. Control myotubes were treated 10% serum from sham mice and 0.01% DMSO).