Interactions among ryanodine receptor isotypes contribute to muscle fiber type development and function

Abstract

Mutations affecting ryanodine receptor (RyR) calcium release channels commonly underlie congenital myopathies. Although these channels are known principally for their essential roles in muscle contractility, mutations in the human RYR1 gene result in a broad spectrum of phenotypes, including muscle weakness, altered proportions of fiber types, anomalous muscle fibers with cores or centrally placed nuclei, and dysmorphic craniofacial features. Currently, it is unknown which phenotypes directly reflect requirements for RyRs and which result secondarily to aberrant muscle function. To identify biological processes requiring RyR function, skeletal muscle development was analyzed in zebrafish embryos harboring protein-null mutations. RyR channels contribute to both muscle fiber development and function. Loss of some RyRs had modest effects, altering muscle fiber-type specification in the embryo without compromising viability. In addition, each RyR-encoding gene contributed to normal swimming behavior and muscle function. The RyR channels do not function in a simple additive manner. For example, although isoform RyR1a is sufficient for muscle contraction in the absence of RyR1b, RyR1a normally attenuates the activity of the co-expressed RyR1b channel in slow muscle. RyR3 also acts to modify the functions of other RyR channels. Furthermore, diminished RyR-dependent contractility affects both muscle fiber maturation and craniofacial development. These findings help to explain some of the heterogeneity of phenotypes that accompany RyR1 mutations in humans.

Keywords: Congenital myopathy; Muscle development; Muscle function; Ryanodine receptors; Zebrafish disease model.

Fig. 1.

Mutant ryr alleles do not produce protein products. (A) Schematic of a cross-section of the trunk of a 1-2 dpf zebrafish embryo highlighting muscle organization. SC spinal cord, noto notochord. (B-D) Transverse cryosections of 24 hpf embryo trunks illustrating the RNA expression patterns of (B) ryr1a, (C) ryr1b, and (D) ryr3 detected by WISH. (E-H″) Transverse sections through the trunks of 48 hpf wild-type and mutant embryos immunostained with the 34C (anti-RyR) antibody. Brightfield images (E-H), fluorescent images showing 34C staining (green) (E′-H′), and merged brightfield/fluorescent images (E″-H″) of embryos of indicated genotypes. Each ryr mutation is associated with loss of a distinct component of the normal expression pattern of RyR channels in embryonic muscle. Somitic muscle of triple ryr1a;ryr1b;ryr3 mutants lack all RyR protein detected by the 34C antibody.

Fig. 2.

Formation of Shh-dependent muscle in ryr mutants. Muscle cell type patterning in wild-type and mutant zebrafish 24 hpf embryos was assessed by immunohistochemical staining for expression of the Prox1 and Engrailed nuclear proteins. (A-D) Representative images of Prox1 (magenta) and Engrailed (green) staining of somitic muscle in wild-type (A), MZryr1a (B), MZryr3 (C) and MZryr1a;MZryr3 (D) embryos. Slow muscle pioneer cells (MPs) were identified as cells that expressed both Prox1 and Engrailed antigens; medial fast fibers (MFFs) were identified as cells that expressed only the Engrailed antigen; and superficial slow fibers (SSFs) were identified as cells that expressed only the Prox1 antigen. (E) Quantification of Shh-dependent muscle cell types. One-way ANOVA was used to determine statistical relationships with Sidak's multiple comparisons test used to adjust P-values. n.s., not significant; *P<0.01, **P<0.001.

Fig. 3.

Single and compound ryr mutants have altered escape velocities. (A) Still images from high-speed recordings of startle response in 48 hpf larvae taken from Movie 1 (wild-type) and Movie 2 (ryr1b). The genotypes of animals are displayed on the left and times after stimulation are displayed along the bottom. The wild-type animal displays characteristic C-bend behavior, whereas the ryr1b mutant never displays a contraction of the entire trunk. (B) Escape velocities of 3 dpf wild-type and mutant larvae. The ryr1b mutants displayed reduced escape velocity, whereas ryr1a;ryr1b double mutants were paralyzed. Loss of RyR1a, RyR3 or both RyR1a and RyR3 channels resulted in larvae that swam faster than wild-type controls. Statistical significance was determined using one-way ANOVA test with Tukey's multiple comparisons test used to adjust the P-values. ns, not significant.

Fig. 4.

Muscle fiber type-specific roles of RyR channels. (A) Schematic for generating zebrafish embryos that mosaically express GCaMP in muscle fibers. One-cell stage embryos were injected with a DNA expression plasmid driving constitutive expression from the B-actin promoter of GCaMP6-slow translationally linked, by a 2A linker peptide, with mCherry. Embryos with isolated muscle fibers that expressed the mCherry reporter were selected for analysis. (B) Experimental setup for SPIM imaging. The 2 dpf embryos, mounted in agarose in a capillary tube, were placed inside an embryo medium-filled chamber. Electrical stimulation to the head was used to evoke a single muscle contraction, and fluorescent GCaMP signal was recorded. (C) GCaMP6-slow signals were recorded from individual fast muscle fibers during a contraction. Loss of RyR1a or RyR3 channels did not alter Ca²⁺ release; however, loss of RyR1b eliminated all Ca²⁺ release in fast fibers. (D) GCaMP6-slow signals were recorded from individual slow muscle fibers during a contraction. Loss of RyR1b activity reduced Ca²⁺ release, whereas loss of RyR1a activity resulted in increased release of Ca²⁺ in slow fibers. Arrows indicate time at which electrical stimulus was delivered. Error bars indicate s.e.m.

Fig. 5.

RyR-mediated muscle contractions are required for slow fiber maturation. (A-I) Slow fibers were visualized with F59 antibody at 20 and 24 hpf and with the slow fiber-specific S58 antibody at 48 hpf. Slow fibers, which are initially wavy, mature and align with respect to each other as wild-type embryos develop. In ryr1b mutant embryos, maturation is delayed; in paralyzed triple mutants, fiber maturation is arrested. (J) Sample image indicating how slow muscle fiber (yellow) and A-P somite (magenta) lengths were determined using ImageJ. (K) Sample image of sarcomere banding indicating how sarcomere A-P lengths (white bracket) were determined. (L) Quantification of slow fiber:somite length ratios. (M) Quantification of sarcomere lengths of slow muscle fibers. One-way ANOVA was used to determine statistical relationships at each developmental time point with Tukey's multiple comparisons test used to adjust P-values. To determine fiber length or sarcomere length, five fibers were examined in each of five different embryos for each condition. ns, not significant.

Fig. 6.

Paralyzed ryr mutants have abnormal craniofacial morphology. Craniofacial features of 6 dpf larvae were visualized following staining with Alcian Blue, which marks cartilage. (A-D) Jaw structures of wild-type larvae: lateral view of intact larva (A), lateral view of stained intact larva (B), isolated upper jaw (C) and isolated lower jaw (D). (E-H) Jaw structures of paralyzed ryr1a;ryr1b;ryr3 larvae: lateral view of intact larva (E), lateral view of stained intact larva (F), isolated upper jaw (G) and isolated lower jaw (H). Jaw architecture of mutant larvae was dramatically shortened in the A-P dimension and wider than normal. Highlighted features include the shape of hypophyseal fenestre (asterisks), the angle between the midline and ceratohyal cartilages of the lower jaw (brackets) and orientation of Meckel's cartilage (arrows).