Subcellular analysis of Ca2+ homeostasis in primary cultures of skeletal muscle myotubes

Specifically targeted aequorin chimeras were used for studying the dynamic changes of Ca concentration in different subcellular compartments of differentiated skeletal muscle myotubes. For the cytosol, mitochondria, and nucleus, the previously described chimeric aequorins were utilized; for the sarcoplasmic reticulum (SR), a new chimera (srAEQ) was developed by fusing an aequorin mutant with low Ca affinity to the resident protein calsequestrin. By using an appropriate transfection procedure, the expression of the recombinant proteins was restricted, within the culture, to the differentiated myotubes, and the correct sorting of the various chimeras was verified with immunocytochemical techniques. Single-cell analysis of cytosolic Ca concentration ([Ca](c)) with fura-2 showed that the myotubes responded, as predicted, to stimuli known to be characteristic of skeletal muscle fibers, i.e., KCl-induced depolarization, caffeine, and carbamylcholine. Using these stimuli in cultures transfected with the various aequorin chimeras, we show that: 1) the nucleoplasmic Ca concentration ([Ca](n)) closely mimics the [Ca](c), at rest and after stimulation, indicating a rapid equilibration of the two compartments also in this cell type; 2) on the contrary, mitochondria amplify 4-6-fold the [Ca](c) increases; and 3) the lumenal concentration of Ca within the SR ([Ca](sr)) is much higher than in the other compartments (>100 ?M), too high to be accurately measured also with the aequorin mutant with low Ca affinity. An indirect estimate of the resting value (~1-2 mM) was obtained using Sr, a surrogate of Ca which, because of the lower affinity of the photoprotein for this cation, elicits a lower rate of aequorin consumption. With Sr, the kinetics and amplitudes of the changes in [cation](sr) evoked by the various stimuli could also be directly analyzed.