Titin Analysis

Titin protein isoforms were analyzed in a subset of animals (n = 4 per group) by SDS-VAGE (Warren et al., 2003). Frozen samples (~30 mg) from the EDL and SOL were homogenized in solubilization buffer [61 mM Tris, 2.78% SDS, 5% 2-B mercaptoethanol, 11% glycerol, 0.02% (w/v), bromophenol blue, and 4 μg/ml leupeptin (pH 6.8)] at a ratio of approximately 0.03 μg muscle mass/μl buffer. Titin standards from rat cardiac (~3,000 kDa) and rat gastrocnemius (~3,600 kDa; Li et al., 2012) were also homogenized and mixed to create a standard cocktail. Samples were incubated for 5 min on ice and then heated for 10 min at 60°C. Protein bands were visualized with a Coomassie G-250 stain. Gels were then digitized and analyzed (FluoraChem HD2 chemiluminescent, Alpha Inotech, Santa Clara, US) for their molecular mass, which was based on the mobility of the experimental bands relative to the rat cardiac and gastrocnemius titin standards. Molecular mass was compared among training groups from the same muscle.