Multi-Pronged Skeletal Muscle Fiber Type Characterization through Immunostaining, Contraction Analysis, and qPCR

Skeletal muscles mediate all voluntary movement and are composed of different fiber types that enable a wide range of functionality by tuning contractile strength and metabolic needs. Muscle fiber types change with physiological and pathophysiological stimuli. To study fiber type transformation in vitro, we need a way to reliably characterize and monitor fiber types. In this study, we optimized a three-pronged strategy to map fiber types in engineered muscle tissues differentiated from murine C2C12 myoblasts: immunostaining, quantitative PCR (qPCR), and analysis of contraction dynamics in response to electrical stimulation. These studies enabled us to understand how varying fiber morphology and gene expression impacts function.

After an initial screening to identify optimal dilutions and combinations of antibodies against specific myosin heavy chain isoforms representative of slow- and fast-twitch fibers, we investigated the changes in fiber type composition throughout the muscle maturation process. We analyzed muscle fibers on day 6, 10, and 14 of differentiation through stimulated contraction and calcium imaging assays, and performed qPCR and immunostaining analysis at the end of the experiment. Overall, the optimization of this three-pronged strategy has enabled better investigation of fiber type changes, with applications ranging from medicine to biohybrid machines.