Calcium transport in intact mammalian fast-twitch and slow-twitch skeletal muscle fibers

Abstract

Calcium ions that are released from the Sarcoplasmic Reticulum (SR) upon electrical stimulation bind to troponin C of the thin filament structure, and initiate contraction of the skeletal muscle fiber. In order for the muscle fiber to relax, calcium must dissociate from troponin C and be removed from the cytoplasm by reuptake via the SR Calcium ATPase or by binding to cytoplasmic proteins. The intricacies of the calcium removal system in intact mammalian fibers have not been elucidated. The goal of my thesis project was to characterize the calcium removal system in mammalian intact skeletal muscle fibers, and determine the contribution of the individual components involved, such as the SR calcium ATPase, troponin C, and parvalbumin. Rat fast-twitch flexor digitorum brevis fibers (FDB) and slow-twitch soleus fibers are enzymatically dissociated and suspended in low melting temperature agarose gel to minimize fiber movement during fluorescence recordings. FDB fibers and soleus fibers are loaded with fura-2 (cell permeant form) and electrically stimulated by 1 to 40 pulses. Florescence signals are recorded at 380 (calcium sensitive) and 358 (calcium insensitive) nm excitation. Ca2+ is calculated assuming non-instantaneous equilibrium with fura-2. The rate constant of calcium decay decreased significantly with increasing stimulation duration in the FDB fibers, but remained relatively constant in the soleus fibers. This is due to expected differences in parvalbumin concentration between fast-twitch and slow-twitch fibers. In fast-twitch fibers parvalbumin becomes increasing saturated by calcium with increasing stimulation durations and can no longer contribute to the decay of calcium. However, there is negligible amounts of parvalbumin in slow-twitch fibers, therefore they do not exhibit this slowing of calcium decay effect. Quantification of the SR calcium ATPase, troponin C and parvalbumin content, using SDS page and immunoblotting techniques confirmed that there was a significant difference in the concentration of parvalbumin between rat FDB (1.2 mM calcium binding site concentration) and soleus fibers ({dollar}<{dollar}50 {dollar}\mu{dollar}M calcium binding site concentration). Unexpectedly there was no significant difference in the concentration of SR calcium ATPase, and troponin C. The values determined by the gel and immunoblot Quantification were well supported by preliminary modeling analysis of the Ca2+ decay. In conclusion, there are significant differences in the decay of Ca2+ in rat fast-twitch and slow-twitch muscle, which is due to differences in parvalbumin concentration. This indicates that parvalbumin has a significant role in the decay of calcium in mammalian skeletal muscle fibers.