An in vivo model for studying the dynamics of intracellular free calcium changes in slow- and fast-twitch muscle fibres

The understanding of the regulation of the free cytosolic [Ca2+] ([Ca2+](i) ) in skeletal muscle is hampered by the lack of techniques for quantifying free [Ca2+](i) in muscle fibres in situ. We describe a model for studying t he dynamics of free [Ca2+](i) in the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus (SOL) muscles of the rat in vivo using caf feine superfusion to induce changes in free [Ca2+](i). We assumed that diff erences in sensitivity between the two muscle types for this substance refl ect differences in intracellular Ca2+ handling in the fibres of which these muscles consist. The Indo-1 ratiometric method, using intravital microscop y with incident light, was adapted to measure free [Ca2+](i) in vivo. Fluor escence images were collected by means of a digital camera. Caffeine superf usion at 37 degrees C for 2 min, at concentrations of 1, 2, 5, 10 or 20 mmo l/l, induced a concentration-dependent increase in free [Ca2+](i) and revea led differences in caffeine sensitivity between the muscle types, with the SOL being more sensitive. In a separate set of experiments the contracture threshold, as assessed by topical application of caffeine, was determined i n both muscle types. EDL had a higher threshold for developing contracture than SOL. These finding are in agreement with previous in vitro studies. We may conclude that the dynamics of free [Ca2+](i) can be assessed reliably in intact mammalian muscle in vivo.