Zebrafishrelatively relaxedmutants have a ryanodine receptor defect, show slow swimming and provide a model of multi-minicore disease

Abstract

Wild-type zebrafish embryos swim away in response to tactile stimulation. By contrast, relatively relaxed mutants swim slowly due to weak contractions of trunk muscles. Electrophysiological recordings from muscle showed that output from the CNS was normal in mutants, suggesting a defect in the muscle. Calcium imaging revealed that Ca 2+ transients were reduced in mutant fast muscle. Immunostaining demonstrated that ryanodine and dihydropyridine receptors, which are responsible for Ca 2+ release following membrane depolarization, were severely reduced at transverse-tubule/sarcoplasmic reticulum junctions in mutant fast muscle. Thus, slow swimming is caused by weak muscle contractions due to impaired excitation-contraction coupling. Indeed, most of the ryanodine receptor 1b (ryr1b) mRNA in mutants carried a nonsense mutation that was generated by aberrant splicing due to a DNA insertion in an intron of the ryr1b gene, leading to a hypomorphic condition in relatively relaxed mutants. RYR1 mutations in humans lead to a congenital myopathy, multi-minicore disease (MmD), which is defined by amorphous cores in muscle. Electron micrographs showed minicore structures in mutant fast muscles. Furthermore, following the introduction of antisense morpholino oligonucleotides that restored the normal splicing of ryr1b, swimming was recovered in mutants. These findings suggest that zebrafish relatively relaxed mutants may be useful for understanding the development and physiology of MmD.

Differences in locomotor performance between individuals: importance of parvalbumin, calcium handling and metabolism

“…The explanation may be that increased DHPR concentrations are important for sprint performance by improving excitation-contraction coupling, but that blocking DHPR mediated Ca 2+ release affects U crit by its depressing effect on cardiovascular performance as outlined above. That both DHPR and RyR are important for sprint performance is supported by the fact that zebrafish relatively relaxed mutants, in which densities of both receptors are severely reduced, have significantly slower sprint velocities (Hirata et al, 2007).…”

“…At a protein level, we predicted that (iii) sprint performance is associated with increased activity of the fast acting ATP-release enzyme creatine kinase (Saks, 2008) and the glycolytic enzyme lactate dehydrogenase, and that sustained performance is linked to greater activity of the mitochondrial enzyme citrate synthase (Seebacher and Glanville, 2010). With respect to Ca 2+ handling proteins, we predicted (iv) that inhibition of DHPR will affect sustained performance negatively, while inhibition of RyR will decrease sprint performance (Hirata et al, 2007;James et al, 2011). Lastly, we tested the hypothesis that (v) increased parvalbumin expression will enhance sprint and sustained locomotion (Arif, 2008;James et al, 2011).…”

Triadopathies: An Emerging Class of Skeletal Muscle Diseases

“…One barrier to treatment development is the relatively small number of preclinical models, and the difficulty presented by the phenotypes of those models. The Ryr1 knockout mouse ("dyspedic") dies at birth, and thus is not suitable for most in vivo studies [38,39]. The available RyR1 "knockin" mouse models, in which identified disease mutations are engineered into the mouse genome, model these conditions well and have provided a wealth of information about MHS, core myopathy, and RyR1 pathophysiology [40].…”

“…In addition, no mouse model exists that recapitulates a non-MH/CCD-related RYR1 myopathy. Recently, the zebrafish model has been utilized as an excellent preclinical vertebrate model for therapy identification [39]. This is well illustrated by the fact that NAC was identified as a potential therapeutic intervention using this system [33].…”

Non–Ca 2+ -conducting Ca 2+ channels in fish skeletal muscle excitation-contraction coupling

“…Recently, two isoforms of the skeletal muscle RyR1 were identified in zebrafish (18), with RyR1a being expressed in superficial slow muscle (also called red muscle), which is aerobic and used for persistent swimming, and RyR1b as the predominant isoform in deep fast muscle (white muscle), the glycolytic musculature used for burst activity. To test if RyR1a and RyR1b might be explicit molecular interaction partners for zf-α 1S -a and zf-α 1S -b, respectively, we performed quantitative RT-PCR on each of these muscle layers, isolated from adult zebrafish.…”