CROOKED NECK DWARF (CN) MUTANT CHICKEN SKELETAL-MUSCLE CELLS IN LOW-DENSITY PRIMARY CULTURES FAIL TO EXPRESS NORMAL ALPHA-RYANODINE RECEPTOR AND EXHIBIT A PARTIAL MUTANT PHENOTYPE

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 s uitable for studies of this mutation. The properties of cn/cn muscle c ells maintained in low density primary cultures (6.25 x 10(3) cells/cm 2) are described in this report. In normal muscle cells, the alpha rya nodine receptor (RyR) isoform appears prior to, and at greater levels than, the betaRyR, and is detected in mononucleated myocytes. The beta RyR isoform appears within 24 hr after the initiation of myotube forma tion, which is earlier than anticipated from studies with intact embry onic muscle. Normal alphaRyR protein is not detected in cultured cn/cn muscle cells, whereas the betaRyR, the alpha1-subunit of the dihydrop yridine receptor, the sarcoplasmic reticulum Ca2+-ATPase, and calseque strin are expressed at comparable levels in normal and mutant muscle c ells. Calcium transients elicited by electrical stimulation, acetylcho line, and caffeine are similar in normal and cn/cn cultured myotubes a nd are blocked by ryanodine in both cell types. In addition, comparabl e L- and T-type calcium currents are observed in normal and mutant mus cle cells, suggesting that both the alpha1-subunit of the dihydropyrid ine receptor and the betaRyR in mutant muscle cells are functional. No rmal and cn/cn muscle cells proliferate and form myotubes in a similar manner. These latter events do not appear to depend on sarcoplasmic r eticulum calcium release, as they also occur in normal muscle cells in which calcium release is prevented by chronic treatment with 100 muM ryanodine. Both cn/cn and ryanodine-treated normal muscle cells exhibi t morphological changes similar to those observed in intact cn/cn skel etal muscle. Thus, the mutant phenotype observed in ovo is partially e xpressed under low density culture conditions, and neither betaRyR pro tein nor its function appear to be capable of preventing the associate d changes. (C) 1993 Wiley-Liss, Inc.