Calcium imaging in C2C12 muscle myocytes to examine the impact of calmodulin mutations on cell signaling
Harris, Brandon J.
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The oxidative stress theory of aging theorizes inadequate redox signaling as a leading cause of aging phenotype. Current studies depict protein oxidation in skeletal muscle as a contributing cause to sarcopenia and other age-related disorders. As a cornerstone in effective muscle contraction, it is expected that calcium handling is altered with age. Calmodulin (CaM), a ubiquitous calcium handling protein, is an effective regulator of calcium via the sarcoplasmic reticulum ryanodine receptor type I. Further, CaM is expected to regulate AMP-dependent protein kinase (AMPK): a whole-body, redox- dependent metabolic regulator. CaM methionine-to-glutamine (MàQ) mutants are emerging as a basis for site-specific oxidation mimic studies. The current study optimizes the efficacy of C2C12 myoblasts and myotubes for non-ratiometric calcium imaging and densitometric AMPK western blot detection. This optimized methodology describes typical considerations for C2C12 calcium imaging and provides context for future studies using M109Q and M124Q CaM mutants. AMPK activity detection requires rigorous optimization and dose-dependent validation before considering CaM MàQ comparisons.