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 the 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 caffeine superfusion to induce changes in free [Ca2+](i). We assumed that differences in sensitivity between the two muscle types for this substance reflect differences in intracellular Ca2+ handling in the fibres of which these muscles consist. The Indo-1 ratiometric method, using intravital microscopy with incident light, was adapted to measure free [Ca2+](i) in vivo. Fluorescence images were collected by means of a digital camera. Caffeine superfusion at 37 degrees C for 2 min, at concentrations of 1, 2, 5, 10 or 20 mmol/l, induced a concentration-dependent increase in free [Ca2+](i) and revealed 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 in 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.