The phenotypic plasticity of skeletal muscle due to changes in functional demand has long been appreciated but the mechanisms involved are poorly defined. In particular, the modulation of key proteins involved with excitation-contraction (EC) coupling by muscle loading condition is largely unexplored. The broad objective of the proposed research is to elucidate how muscle loading condition modulates components of EC coupling. These components include a synaptic protein involved with membrane excitation (nicotinic acetylcholine receptor), a protein in the T-tubule involved with Ca2+ release (dihydropyridine receptor), and a sarcoplasmic reticulum Ca2+ reuptake protein (Ca 2+ ATPase pump). Muscle loading condition will be altered by either unweighting the rat hindlimb (unloading), returning unloaded rats to weightbearing (reloading), surgical ablation of hindlimb synergistic muscles to increase muscle loading (overloading), or muscle denervation to inhibit neuromuscular activation and thereby active muscle loading.
The specific aims are: (1) To determine the modulation of mRNA and protein expression of EC coupling components over a course of muscle unloading, reloading, overloading, and denervation; (2) To correlate contractile outcomes to gene expression of EC coupling proteins; (3) To determine if neuromuscular activation is a triggering mechanism for the adaptive responses to changes in muscle loading condition. Messenger RNA levels will be measured by Northern blot analysis and protein levels by SDS-PAGE and Western blot analysis. Transcriptional activation will be measured by nuclear run-off assay. Contractile properties will be measured in vitro at 25 degrees C. Since changes in muscle loading condition may also invoke changes in neuromuscular activity, measurement of the muscle activation status by electromyography, using indwelling muscle electrodes, during different loading conditions will help to establish more precise links between neuromuscular activity and nuclear events. Hypotheses will be tested that shifts in molecular outcomes will be correlated with physiological changes. Concepts about the regulation of muscle proteins by loading can be applied toward understanding, preventing, and recovering from muscle weakness and atrophy due to sedentary lifestyles, exposure to spaceflight, and inactivity secondary to disease, and to the improvement of muscle function for work performance.
