The spatiotemporal characteristics of the Ca2+ release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 μM of the low-affinity Ca2+ indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca2+ chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca2+] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks (∼23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca2+ transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest ΔF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca2+ release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in ∼1.4 ms and dissipate within ∼4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca2+ transients peaked at T-tubule locations, was 0.62 μm, similar to the 0.61 μm measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca2+ domains in adult mammalian muscle fibers are confined to Ca2+ release sites located at the junctional sarcoplasmic reticulum (SR).
Quantification of Mitochondrial Calcium Dynamic Changes During Voltage-Induced Calcium Release in Mammalian Skeletal Muscle