Calcium release induced by high K+ and caffeine in cultured skeletal muscle cells of embryonic chicken

Abstract

To further understand the function of excitation-contraction coupling in skeletal muscle cells developing in vitro, Ca2+ transients elicited by high-K+ depolarization in the presence and absence of extracellular Ca2+ were compared with Ca2+ release induced by caffeine in cultured skeletal muscle cells isolated from 9-day-old chicken embryos (E9). Almost all myoblasts and myotubes cultured for 1 (E9I1) to 8 (E9I8) days responded to 80 mM [K+]O with an elevation of [Ca2+]i. Although all myotubes cultured for more than 4 days exhibited Ca2+ release independent of extracellular Ca2+, only about 50% of E9I1 and E9I2 cells maintained their response to Ca(2+)-free high-[K+]O solution. Strikingly, a considerable proportion of cells of short-term culture were insensitive to 10 mM caffeine. Moreover, 46.8% of the caffeine-insensitive E9I1 and E9I2 cells, 29 out of 62, was still responsive to 80 mM [K+]O in the absence of extracellular Ca2+. Western blot and immunocytochemistry showed that ryanodine receptor (RyRs) expression increases with culture. The Ca2+ release from caffeine-insensitive cells induced by Ca(2+)-free high-[K+]O solution could be blocked by 100-200 microM ryanodine, which suggests the involvement of RyRs. Evidence is presented to show that a low resting [Ca2+]i may be one factor responsible for the caffeine insensitivity of RyRs in cells of short-term culture.

Physiological and ultrastructural features of human induced pluripotent and embryonic stem cell-derived skeletal myocytes in vitro

“…This is in line with previous reports on skeletal muscle EC coupling using either K + -induced depolarization or electric stimulation (27)(28)(29). The initial, relatively fast decay phase likely corresponds to the binding of cytosolic Ca 2+ to fast Ca 2+ buffers (troponin) (30).…”

“…In contrast, the slow decay phase displayed a higher variability for both cell types and slower rates in iPS-myocytes. Interestingly, it has been shown that the slow component of the intracellular Ca +2 decay becomes faster with vertebrate development (27)(28)(29) (31). Taken together, our data suggest that the slow Ca 2+ buffering processes that follow muscle contraction may still remain immature under these culture conditions in both cell types and to a higher extent in iPS-myocytes.…”

“…Therefore, the occurrence of a biphasic change in [Ca 2+ ]i upon K + -induced membrane depolarization in the absence of extracellular Ca 2+ provides evidence for a skeletal-musclespecific EC coupling in ES-and iPS-myocytes, and is in line with the presence of skeletal-muscle L-type Ca 2+ currents. The rate of rise in [Ca 2+ ]i, which reflects the rate of Ca 2+ release, showed a nonuniform distribution, suggesting various degrees of differentiation for EC coupling in both cell types, reminiscent of what has been reported in developing skeletal muscle (27)(28)(29). Nonetheless, the fastest rates of release (lowest dt values), which should reflect more mature EC coupling, concerned the majority of cells.…”

Triadin (Trisk 95) Overexpression Blocks Excitation-Contraction Coupling in Rat Skeletal Myotubes

“…4A) on control, T95-myotubes, or on T51-myotubes by changing to 140 mM KCl. It resulted, as expected (27)(28)(29), in an increase in intracellular calcium concentration followed by a return to the basal level in control myotubes as well as in T51-myotubes. Nevertheless, in T95-myotubes, this effect was almost abolished, and depolarization induced only a minor calcium release (Fig.…”