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Intracellular calcium (Ca(2+)) signals are essential for several aspects of muscle development, including myofibrillogenesis-the terminal differentiation of the sarcomeric lattice. Ryanodine receptor (RyR) Ca(2+) stores must be operative during this period and contribute to the production of spontaneous global Ca(2+) transients of long duration (LDTs; mean duration approximately 80 s). In this study, high-speed confocal imaging of intracellular Ca(2+) in embryonic myocytes reveals a novel class of spontaneous Ca(2+) transient. These short duration transients (SDTs; mean duration approximately 2 s) are blocked by ryanodine, independent of extracellular Ca(2+), insensitive to changes in membrane potential, and propagate in the subsarcolemmal space. SDTs arise from RyR stores localized to the subsarcolemmal space during myofibrillogenesis. While both LDTs and SDTs occur prior to myofibrillogenesis, LDT production ceases and only SDTs persist during a period of rapid sarcomere assembly. However, eliminating SDTs during this period results in only minor myofibril disruption. On the other hand, artificial extension of LDT production completely inhibits sarcomere assembly. In conjunction with earlier work, these results suggest that LDTs have at least two roles during myofibrillogenesis-activation of sarcoplasmic regulatory cascades and regulation of gene expression. The distinct spatiotemporal patterns of LDTs versus SDTs may be utilized for differential regulation of cytosolic cascades, control of nuclear gene expression, and localized activation of assembly events at the sarcolemma.
Fig. 2. Peripheral distribution of RyRs coincident with SDTs and myofibrillogenesis. (A) A maximum projection of 90 confocal sections (250 nm steps) from a 34Cstained
myocyte at 48 h in culture. Note the sarcomeric distribution of RyRs throughout the cell; spherical objects outside the cell are non-specific staining of free
yolk inclusions. The horizontal and vertical lines indicate the plane of section for C and D, respectively. Scale bar is 10 Am. (B) A single z slice at 5.5 Am from the
substrate (cell maximum z is 13.5 Am) shows how RyRs are restricted to the subsarcolemmal space. Both RyR stores and sarcomeric proteins are restricted to this
space during SDT production and myofibrillogenesis during the first 48 h in culture. Vertical line is the plane of section for the image shown in E. (C) Avertical slice
along the x axis shows the same pattern as seen in B. This peripheral distribution of RyRs was observed in all myocytes with immature (not shown) or mature striated
34C staining patterns. Vertical scale bar is 10 Am. (D) Cross-section at the location indicated in A. Vertical line represents substrate location in D and E (that is, top of
cell is to left). (E) Cross-section at the location indicated in B.
Fig. 6. SDTs are not required for sarcomere assembly. Normal organization of actin (A, phalloidin staining) and myosin (B, MF20 staining) into sarcomeric arrays at 48
h in culture. Note the tight longitudinal myofibril bundles and transverse alignment of sarcomeres. Scale bar in B is 10 Am. When ryanodine [100 AM] is applied from 17
to 24 h in culture, sarcomere assembly is slightly reduced (C, D). Note that some sections of myofibrils become unbundled and deviate from the longitudinal axis,
sometimes as much as 90- as seen in this cell. Scale bar inDis 10 Am. (E) Sarcomeric actin and myosin assembly in the presence of ryanodine [100 AM] from 17 to 24 h in
culture. Cells were fixed and examined at either 24 h in culture or ryanodine was washed out at 24 h and cells were allowed to develop until fixation at 48 h in culture.
Note the moderate decrease in sarcomeric organization at 24 h and that, despite ryanodine washout, assembly does not recover in the following 24 h period. (F) Later
application of ryanodine does not affect sarcomere assembly. Note in comparing columns that a 4 h blockade from 20 to 24 h has no effect on myofibrillogenesis (*P <
0.01; **P < 0.0001).
