Silencing RyR3 Eliminates Parajunctional Feet and Ca Sparks in Zebra Fish Tail Myotomes

All skeletal muscles express similar levels of RyR1, but the level or RyR3 expression varies greatly. Anatomically, this correlates with two types of “feet” (= RyR): junctional feet (JF), located in the triad gap of all muscles, and parajunctional feet (PJF), located on the sides of the jSR cisternae and present only in RyR3-expressing muscles. Physiologically, frog fibers (with a high content of RyR3 and PJF) have readily-detected Ca sparks, while adult mouse fibers (with a very low or nil RyR3 and PJF content) do not. Fast fibers in zebrafish tail display abundant PJF. We explored the role of RyR3 by injecting zebrafish oocytes with a splicing blocker morpholino designed to bind to RyR3 pre-mRNA. Tails from several fish at 72 hpf (hours post fertilization) were pooled for western blotting. A pan-RyR antibody (34C) reveals three bands, presumably representing RyR3 and RyR1a and b. The slowest band specifically disappears after morpholino injection. Tails from ∼50 larvae at 72 hpf were collected for enzymatic dissociation of single muscle fibers. Intact 48 and 72 hpf tails and some dissociated fibers were fixed for EM. The PJF/JF ratio in EM images of fast fibers in control larvae at 72 hpf was 0.79±0.14, mean±SD (7 fish, 70 fibers, 310 triads; 3 experiments) and decreased to 0.03±0.03 (10 fish, 100 fibers, 420 triads; 4 experiments) in injected larvae. To measure Ca sparks, dissociated fibers were adhered to coverslips by matrigel, loaded with Fluo-4, and imaged confocally. 0.3 mM caffeine was used to stimulate sparks, which were readily detected in normal cells but almost absent in morpholino-treated cells. These data identify PJF as RyR3 and indicate that RyR3 activity is required for the ready-detection of sparks.