Mybphb acts as a molecular brake in the C-zone. (A) Abundance of MyBP isoforms in fast-twitch muscle samples from adult wildtype and mybpc2b^−/− zebrafish relative to total myosin heavy chain. A homozygous null mutation in mybpc2b (SA10810) ablates Mybpc2b and increases the abundance of Mybphb, maintaining a consistent molar ratio of MyBPs to myosin heavy chains observed in the wiltype zebrafish. (B–D) The sliding of fluorescently labeled actin filaments along native myosin thick filaments isolated from wildtype and mybpc2b^−/− adult zebrafish fast muscle. (B) An illustration of wildtype and mutant thick filaments with C-zones colored to represent MyBP isoform content as in A. The native thick filament assay observes the movement of a fluorescently labeled actin filament being propelled over the thick filament surface by the myosin heads emanating from the thick filament. Actin filament displacement trajectories have an initial fast phase associated with the D-zone, which is devoid of MyBP, followed by a potential slower phase as it moves over the MyBP within the C-zone. (C) Actin filament displacement versus time data for 13 different actin filaments moving over different wildtype fast-twitch muscle thick filaments demonstrating dual-phase trajectories as illustrated in B with the fast velocity phase (grey data) and slow velocity phase (green data) identified. (D) The fast (grey) and slow (green) velocity phases for actin filament motion over wildtype thick filaments containing a mix of Mybphb and Mybpc2b (74 thick filaments from n = 4 animals) as in A and mybpc2b^−/− thick filaments containing exclusively Mybphb (116 thick filaments from n = 3 animals). Statistics were calculated using a two-tailed Student’s t test. ns denotes P > 0.05. Data are presented as means ± 1 SD.