Hereditary Pituitary Dwarfism in Mice Affects Skeletal Cardiac Myosin Isozyme Transitions Differently

The dwarf mutation in mice interferes with the development of those anterior pituitary cells responsible for production of thyroid stimulating hormone, growth hormone, and prolactin. Myosin isozyme transitions in both cardiac and skeletal muscle were also found to be affected in this mutant. Electrophoresis of native myosins demonstrated that the fetal (V3) to adult (V1) ventricular cardiac isozyme transition was completely blocked in dwarf mice; in contrast, the neonatal to adult fast myosin transition in hind limb skeletal muscle was slowed but not totally inhibited. The persistence of neonatal myosin heavy chain for up to 55-75 d after birth in dwarf mice, as compared with 16 d in normal mice, was directly demonstrated by polypeptide and immunopolypeptide mapping. Morphological examination of 18-36-dold dwarf skeletal muscles by optical and electron microscopy revealed a relative immaturity, but no signs of gross pathology were evident. Immunocytochemical analysis showed that the abnormal persistence of neonatal myosin occurs in most of the fibers. Multiple injections of thyroxine restored a normal isozyme complement to both cardiac and skeletal muscles within 11-15 d. Therefore, the effects of the dwarf mutation on myosin isozymes can be explained by the lack of thyroid hormone in these animals. Because the synthesis of growth hormone is not stimulated by thyroid hormone in dwarf mice as it would be in normal animals, these results demonstrate that thyroid hormone promotes myosin isozyme transitions independent of growth hormone production. Six or more different isoforms of the myosin heavy chain can be found in mammalian cardiac and skeletal muscles. Although their structures are very homologous, these heavy chains are distinct polypeptides as shown by protein chemical, immunochemical, and molecular cloning approaches (5, 14, 16, 19, 20, 25, 29, 35, 41, 43, 44, 47). In both developing and adult muscles, transitions between different myosin isoforms take place within the same cardiac cells or skeletal muscle fibers (7, 8, 32, 34). The control of these transitions has in several cases been shown to be modulated by factors external to the muscle cell, although the nature of these controlling influences is known in general outline only (reviewed in reference 42). Some myosin transitions require a specific innervation or nerve-activity pattern (23, 33), whereas others can occur in the absence ofinnervation (8, 17). The hormonal status of the animal can also influence the type of myosin isozyme present. Hypothyroidism has been shown to inhibit certain myosin isozyme transitions that normally take place during roamTHE JOURNAL OF CELL BIOLOGY . VOLUME 101 AUGUST 1985 603-609 © The Rockefeller University Press . 0021-9525185/08]0603/07 $1.00 malian skeletal and cardiac muscle development (9, 13, 17, 30). However, thyroid hormone levels also contribute to the regulation of the postnatal increase in growth hormone (36), and growth hormone has effects on skeletal growth, at least some of which are mediated via the action of somatomedins (27). It is therefore not clear from previous results whether the inhibition of myosin transitions produced by hypothyroidism is a result of the lack of thyroid hormone, or whether it is a secondary effect due to lowered growth hormone levels. To investigate this question, we have studied myosin isozyme transitions in a mouse model of pituitary dwarfism. The dwarf mutation in mice (39) interfers with the development of those anterior pituitary cells that normally produce thyroid stimulating hormone (TSH), t growth hormone (GH), and prolactin (4). As a result, these hormones are present at low levels in the sera of homozygous dwarf mice. In addition, ~Abbreviations used in this paper: GH, growth hormone; TSH, thyroid stimulating hormone. 603 on N ovem er 6, 2017 jcb.rress.org D ow nladed fom synthesis of GH is undetectable in the pituitary at all ages (12, 37, 38), and exogenous thyroid hormone does not stimulate GH production (37). Dwarf mice are therefore a suitable model of the hypothyroid state that is not complicated by the stimulatory effects of thyroid hormone on GH production. We have found that the normal developmental appearance of the adult cardiac myosin isozyme is inhibited in these animals. In contrast, accumulation of the adult skeletal muscle myosin forms is slowed but not completely blocked. Both of these developmental defects can be overcome by exogenous thyroxine. Since dwarf mice lack GH-producing somatotropes (12, 31, 38), these results demonstrate that thyroid hormone does not affect myosin isozyme transitions by stimulating growth hormone production. MATERIALS AND METHODS Mice of the Snell dwarf strain (4, 12) were used in this study. Optical and electron microscopy of fixed extensor digitorum longus muscles was performed using standard techniques (15) on dwarf and control mice of various ages, as follows: 18 d (five dwarfs and three controls), 24-25 d (six dwarfs and eight controls), and 31-36 d (three dwarfs and four controls). For electrophoresis of native myosin, muscle tissue from individual mice was extracted and prepared as described (7); the skeletal muscle extracts were made from the gastrocnemius muscles. Myosin was purified (5) using hind leg muscles pooled from several mice for a given age point. The myosins were denatured with SDS and cleaved with chymotrypsin as described previously (5, 43). The resulting polypeptides were separated on duplicate SDS-containing 10% polyacrylamide gels. One gel was stained with Coomassie Blue and the other was blotted onto a nitrocellulose sheet as described (6), except that 0.1% SDS was included in the buffer used for the electrophoretic transfer step and electrophoresis was carded out for 1820 h. The nitrocellulose sheet was then treated and reacted with an antibody specific to rat neonatal myosin as described (7). Indirect immunofluorescence on gastrocnemius muscles was performed using rabbit antibodies to rat fast and neonatal myosins, prepared and characterized as described (7); a rhodamine-conjugated goat anti-rabbit immunoglobulin preparation (Nordic, Tilburg, The Netherlands) was used as a second antibody. For the hormone supplementation experiments, dwarf mice o f 5 g body wt were injected every 2-3 d with 1 ug L-thyroxine (Sigma Chemical Co., St. Louis, MO) dissolved in 0.1 ml of 0.14 M NaCI, 10 mM sodium phosphate, pH 8.5. After 11-15 d of treatment, the native myosin isozymes were analyzed by electrophoresis.