Crooked Neck Dwarf (cn) mutant chicken skeletal muscle cells in low density primary cultures fail to express normal α ryanodine receptor and exhibit a partial mutant phenotype

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Abstract
The Crooked Neck Dwarf (cn) mutation in chickens causes marked changes in intact embryonic skeletal muscle. We have investigated whether the cn/cn phenotype develops in vitro, and if cultured muscle cells are suitable for studies of this mutation. The properties of cn/cn muscle cells maintained in low density primary cultures (6.25 × 103 cells/cm2) are described in this report. In normal muscle cells, the α ryanodine receptor (RyR) isoform appears prior to, and at greater levels than, the βRyR, and is detected in mononucleated myocytes. The βRyR isoform appears within 24 hr after the initiation of myotube formation, which is earlier than anticipated from studies with intact embryonic muscle. Normal αRyR protein is not detected in cultured cn/cn muscle cells, whereas the βRyR, the α1-subunit of the dihydropyridine receptor, the sarcoplasmic reticulum Ca2+-ATPase, and calsequestrin are expressed at comparable levels in normal and mutant muscle cells. Calcium transients elicited by electrical stimulation, acetylcholine, and caffeine are similar in normal and cn/cn cultured myotubes and are blocked by ryanodine in both cell types. In addition, comparable L- and T-type calcium currents are observed in normal and mutant muscle cells, suggesting that both the α1-subunit of the dihydropyridine receptor and the βRyR in mutant muscle cells are functional. Normal and cn/cn muscle cells proliferate and form myotubes in a similar manner. These latter events do not appear to depend on sarcoplasmic reticulum calcium release, as they also occur in normal muscle cells in which calcium release is prevented by chronic treatment with 100 μM ryanodine. Both cn/cn and ryanodine-treated normal muscle cells exhibit morphological changes similar to those observed in intact cn/cn skeletal muscle. Thus, the mutant phenotype observed in ovo is partially expressed under low density culture conditions, and neither βRyR protein nor its function appear to be capable of preventing the associated changes.