Abstract

The ryanodine receptor (RyR) 1 isoform of the sarcoplasmic reticulum (SR) Ca2+ release channel is an essential component of all skeletal muscle fibers. RyR1s are detectable as "junctional feet" (JF) in the gap between the SR and the plasmalemma or T-tubules, and they are required for excitation-contraction (EC) coupling and differentiation. A second isoform, RyR3, does not sustain EC coupling and differentiation in the absence of RyR1 and is expressed at highly variable levels. Anatomically, RyR3 expression correlates with the presence of parajunctional feet (PJF), which are located on the sides of the SR junctional cisternae in an arrangement found only in fibers expressing RyR3. In frog muscle fibers, the presence of RyR3 and PJF correlates with the occurrence of Ca2+ sparks, which are elementary SR Ca2+ release events of the EC coupling machinery. Here, we explored the structural and functional roles of RyR3 by injecting zebrafish (Danio rerio) one-cell stage embryos with a morpholino designed to specifically silence RyR3 expression. In zebrafish larvae at 72 h postfertilization, fast-twitch fibers from wild-type (WT) tail muscles had abundant PJF. Silencing resulted in a drop of the PJF/JF ratio, from 0.79 in WT fibers to 0.03 in the morphants. The frequency with which Ca2+ sparks were detected dropped correspondingly, from 0.083 to 0.001 sarcomere(-1) s(-1). The few Ca2+ sparks detected in morphant fibers were smaller in amplitude, duration, and spatial extent compared with those in WT fibers. Despite the almost complete disappearance of PJF and Ca2+ sparks in morphant fibers, these fibers looked structurally normal and the swimming behavior of the larvae was not affected. This paper provides important evidence that RyR3 is the main constituent of the PJF and is the main contributor to the SR Ca2+ flux underlying Ca2+ sparks detected in fully differentiated frog and fish fibers.