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

Abstract

Specifically targeted aequorin chimeras were used for studying the dynamic changes of Ca2+ 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 Ca2+ 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 Ca2+ concentration ([Ca2+]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 Ca2+ concentration ([Ca2+]n) closely mimics the [Ca2+Ic, 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 [Ca2+]c increases; and 3) the lumenal concentration of Ca2+ within the SR ([Ca2+Isr) is much higher than in the other compartments (>100 JIM), too high to be accurately measured also with the aequorin mutant with low Ca2+ affinity. An indirect estimate of the resting value (-1-2 mM) was obtained using Sr2+, a surrogate of Ca2+ which, because of the lower affinity of the photoprotein for this cation, elicits a lower rate of aequorin consumption. With Sr2 , the kinetics and amplitudes of the changes in [cationii]sr evoked by the various stimuli could also be directly analyzed. INTRODUCTION Stimulus-contraction (MacLennan et al., 1985;Lytton and MacLennan, 1988;Burk et al., 1989) and the overall molecular architecture of the pump (Toyoshima et al., 1993) as well as the kinetic details of the transport mechanism (Inesi et al., 1992) have been clarified. Similarly, the Ca2+ release channels, also known as the ryanodine receptors (Takeshima et al., 1989;Otsu et al., 1990;Giannini et al., 1992;Sorrentino and Volpe, 1993) (Rizzuto et al., 1992). Through the addition to the cDNA coding for the native photoprotein of specific targeting sequences or through its fusion with suitable polypeptides, we have produced a series of new aequorin chimeras targeted to different intracellular locations, the mitochondrial matrix (Rizzuto et al., 1992, nucleoplasm (Brini et al., 1993nucleoplasm (Brini et al., , 1994 (Franzini-Armstrong et al., 1987) and is thought to play a major role in intralumenal Ca2' binding (Ikemoto et al., 1989;Lytton and Nigam, 1992;Damiani and Margreth, 1994 Primary cultures of skeletal muscle were prepared from newborn rats (2-3 days) as described previously (Cantini et al., 1994). In brief, posterior limb muscles were remove...

High glucose induces adipogenic differentiation of muscle-derived stem cells

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Weight gain related to treatment with atypical antipsychotics is due to activation of PKC-β

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