Contribution of ryanodine receptor type 3 to Ca2+ sparks in embryonic mouse skeletal muscle

The kinetic behavior of Ca2+ sparks in knockout mice lacking a specific rya nodine receptor (RyR) isoform should provide molecular information on funct ion and assembly of clusters of RyRs. We examined resting Ca2+ sparks in Ry R type 3-null intercostal myotubes from embryonic day 18 (E18) mice and com pared them to Ca2+ sparks in wild-type (wt) mice of the same age and to Ca2 + sparks in fast-twitch muscle cells from the foot of wt adult mice. Sparks from RyR type 3-null embryonic cells (368 events) were significantly small er, briefer, and had a faster time to peak than sparks from wt cells (280 e vents) of the same age. Sparks in adult cells (220 events) were infrequent, yet they were highly reproducible with population means smaller than those in embryonic RyR type 3-null cells but similar to those reported in adult amphibian skeletal muscle fibers. Three-dimensional representations of the spark peak intensity (Delta F/Fo) vs. full width at half-maximal intensity (FWHM) vs. full duration at half-maximal intensity (FTHM) showed that wt em bryonic sparks were considerably more variable in size and kinetics than sp arks in adult muscle. In all cases, tetracaine (0.2 mM) abolished Ca2+ spar k activity, whereas caffeine (0.1 mM) lengthened the spark duration in wt e mbryonic and adult cells but not in RyR type 3-null cells. These results co nfirmed that sparks arose from RyRs. The low caffeine sensitivity of RyR ty pe 3-null cells is entirely consistent with observations by other investiga tors. There are three conclusions from this study: i) RyR type-1 engages in Ca2+ spark activity in the absence of other RyR isoforms in RyR type 3-nul l myotubes; ii) Ca2+ sparks with parameters similar to those reported in ad ult amphibian skeletal muscle can be detected, albeit at a low frequency, i n adult mammalian skeletal muscle cells; and iii) a major contributor to th e unusually large Ca2+ sparks observed in normal (wt) embryonic muscle is R yR type 3. To explain the reduction in the size of sparks in adult compared to embryonic skeletal muscle, we suggest that in embryonic muscle, RyR typ e 1 and RyR type 3 channels cc-contribute to Ca2+ release during the same s park and that Ca2+ sparks undergo a maturation process which involves a dec rease in RyR type 3.