The pituitary-muscle axis in mdx dystrophic mice

JOURNAL OF THE

NEU~~CAL SCIENCES ELSEVIER

Journal of the Neurological Sciences 123 (1994) 80-87

. . . . . .

The pituitary-muscle axis in mdx dystrophic mice J.E. Anderson ~'*, L. Liu b,c, E. Kardami
Abstract

As myogenesis, muscle growth and differentiation and growth factor expression are influenced by thyroid and growth hormone (GH) levels, it is important to investigate the possibility that altered activity of the pituitary-muscle axis prevents the lethal progression of rndx dystrophy and/or contributes to the muscle fiber hypertrophy of limb muscles. The ultrastructure of pituitary and thyroid tissues in age-matched control and mdx mice at 2 and 12 months of age was examined. Pituitary GH, and serum thyroid stimulating hormone (TSH), thyroid hormone (T4), and creatine kinase (CK) levels were measured. Mdx thyroid gland structure was similar to age-matched control glands. Mdx thyroid gland weighed significantly more than in age-matched controls, but was unchanged relative to body weight. TSH and T4 levels were not different from levels in control mice. ttigh CK levels reflected the active dystrophy in rndx muscles. Somatotrophs in mdx pituitaries were hypertrophied in comparison to controls, indicating increased secretory activity, and pituitary GH was slightly but significantly greater in old mdx female mice compared to age-matched female controls. These observations rule out hypopituitary or hypothyroid function as a reason for the low impact of dystrophin deficiency in rndx muscles. Results suggest a contribution by raised GH to the fiber hypertrophy in mdx limb and heart muscle, which might also assist the large capacity for limb muscle regeneration in rmtx mice. Key words: mdx mouse; Pituitary; Growth hormone; Thyroid hormone; Muscle regeneration; Hypertrophy

I. Introduction

Thyroid hormone (TH) levels influence muscle and its development at a number of levels. T H promotes terminal differentiation of neonatal muscle (D'Albis et al. 1987). As well, maturation of the sarcoplasmic reticulum Ca 2+ transport is critically dependent on TH, independent of growth hormone (Simonides and van Hardeveld 1989). T H levels affect myosin heavy chain gene expression in rat cardiac and skeletal muscles in a muscle-specific manner (Izumo et al. 1986, 1988), as are isoform transitions during development (Draeger et al. 1987), also independent of G H (Whalen et al. 1985). Growth hormone ( G H ) also has important positive influences on promoting growth of normal skeletal (Ullman and Oldfors 1989) and cardiac muscle (Guler

* Corresponding author. Tel.: (204) 788-6716; Fax: (204) 788-6763. 0022-510X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-5 1 0 X ( 9 3 ) E 0 2 6 9 - F

et al. t988), and on myotubc growth during muscle regeneration (Ullman et al. 1989) likely via GH-indcpendent expression of G H receptor m R N A (Jennische and Andersson 1991). Early myoblast proliferation during development (Ullman and Oldfors 1989, 1991) and muscle regeneration are also promoted by G H , independent of muscle insulin-like growth factor ( I G F - I ) (Sommerland et al. 1989) which is itself G H - d e p e n d e n t (Murphy et al. 1987). Thyroid hormone interaction with G H is debated (Nwoye et al. 1982: Butler-Browne and Whalen 1984; Whaten c t a l . 1985: Simonides and van Hardeveld 1989), likely as interpretation is complicated by interaction of pituitary hormones with glucocorticoids (Orlowski and Lingrel 1990), somatomedins and IGF-I, in hypophysectomized and normal animals (Guler et al. 1988; Isgaard et al. 1989: G r a n t et al. 1991). The effects of body growth and hormone balance on human D M D are uncertain. Two case reports deal

.I E. Anderson et al. / Journal ~/'the Neurological Science 123 (1994) 80-87

with mitigation of DMD in a growth hormone deficient patient (Zatz et al. 1981: Zatz and Frota-Pessoa 1981), and slightly less severe DMD in one identical twin treated with a growth hormone inhibitor (Zatz et al. 1986). A low human GH response to arginine in DMD gave somc evidence of altered hypothalamic-hypophyseal function (Zaccaria et al. 1989). Muscle maturation in Duchenne muscular dystrophy (DMD) is altered. Fetal myosins (Schiaffino et al. 1986) and immature transitional fiber types appear in fibers which regenerate after dystrophic damage (Nonaka et al. 1<)81). Experiments on animals with autosomally-transmitred dystrophic muscle have shown that retardation of myofiber growth or activity by pituitary ablation, dcncrvation (Karpati et al. 1985) or pituitary dwarfism (qotsuka ct al. 1981) prevent muscle fiber necrosis in dystrophy. The effects of hypophysectomy on dystrophy in hamsters may also be related to the reduced my_ ofibcr size in growth hormone-depleted animals (Karpati and Carpenter 1986). In X-linked dystrophindeficient md~ mousc dystrophy, small fibers also appear to be spared from the dystrophic sequelae observed in limb muscles with larger fibers (Karpati et al. 1988). Together these human and animal studies suggest the possibility that the extent of myofiber growth and maturity may contribute to the outcome of muscular dystrophy, possibly through the influence of pituilar-y and thyroid hypofunction on myogenesis, maturation, or growth of regenerating myofibers. .~/dx mouse muscle is genetically dystrophin-deficicnt (Hoffman et al. 1987; Anderson et al. 1990) similar to DMD muscle (Hoffman et al. 1987). However, mdx dystrophy parallels only the onset, X-linkage, and early regeneration of myofibers seen in human DMD, since most of the mdx mouse skeletal muscles largely show a large degree of functional and structural recovery (Anderson ct al. 1987, 1988; Coulton ct al. 1988). Their repair includes both whole muscle and individual fiber hypertrophy (Anderson et aI. 1987, 1988, 1994). Although the favorable prognosis of mdx dystrophy and the apparently large capacity of mdv muscle precursors to repair injured muscle (Zacharias and Anderson 1991; Anderson 1991; Mclntosh ct al. 1994) may involve basic fibroblast growth factor (Anderson et al. 1991. 1993), the mechanisms that promote that recovery are not well understood. To investigate whether alterations of the pituitarymuscle axis could influence the expression and outcome of mdx dystrophy+ the ultrastructure of organcllcs involved with hormone synthesis in pituitary and thyroid glands was compared to controls. In addition. creatinc kinase (CK), thyroid stimulating hormone (TSH), GH, and TH (T4) were measured, as screens for active muscle damage, and pituitary and thyroid gland function.

81

2. Materials and methods

Animals Dystrophic mdx mice (C57BL/10ScSn mdx) and normal controls (C57BL/10ScSn) were bred and housed according to the Canadian Council on Animal Care in the University of Manitoba Central Animal Care Facility. At 2 or 12 months of age, animals were anesthetized (ketamine/rompun, 0.03 ml i.p./20 g body weight). Rapid thoracotomy exposed the heart, and blood was drawn by cardiac puncture into heparinized syringes, transferred into vacutainer tubes (Becton Dickinson and Co., Rutherford, N J), and centrifuged. Serum was pipetted into microcentrifuge tubes, capped and frozen at - 7 0 ° C until assay. Serum samples for TH assay were collected from a total of 55 animals at a variety of ages during the course of other experiments. Samples of blood were collected at sacrifice from 8 control and 6 mak mice at 2 months of age, for commercial TSH assay. All blood samples were coded in order of collection, and decoded only after each assay. Tissue preparation Tissues were prepared for ultrastructural study from animals at 2 and 12 months of age. The entire thyroid gland was removed from under the strap and sternocleidomastoid muscles, weighed, cut into l-ram 3 blocks which were fixed in 3% glutaraldehyde in 0.1 M Sorenson's phosphate buffer (pH 7.35) for 3 h. For electron microscopy, the pituitary gland in the sella turcica was exposed, as reported previously (Anderson and Thliveris 1979), and freed from the fossa by careful dissection. The two lobes of the adenohypophysis (pale) were cut from the midline neurohypophysis. Adenohypophysis blocks were fixed for 4 h by immersion in the same glutaraldehyde fixative. The tissue blocks from six 2-month-old and four 12-month-old animals from each strain were coded to minimize observer bias, and processed for routine electron microscopy as previously reported (Anderson 1991). Eight microscope sections (0.5-/,tin thickness) were stained with toluidine blue, and viewed and photographed using an Olympus BH2 photomicroscope. Electron microscope sections were examined and photographed in a blinded fashion on a Philips 201 microscope. Pituitaries for growth hormone assay were carefully and quickly dissected from a third set of animals (final number: 43), snap frozen, placed in preweighed, numbered microcentrifuge tubes, and stored at - 7 0 ° C until assay. Pituitaries from male and female, control and mdx mice were sampled according to age (old: 8-10 months of age; young: 2 months old). Each vial was weighed prior to growth hormone extraction to determine pituitary weight.

82

.I.E. Anderson et al. / Journal

(if' the Neurological Science 123 (1994) 80-87

Serum TH, TSH, and CK assay Serum T4 level was determined using a colorimetric assay, E n z y m u n - T e s t T4 l m m u n o d i a g n o s t i c s kit (Boehringer Mannheim G m b H Diagnostica, Germany) and read on a spectrophotometer (420 nm). T3 was not measured due to instability before accumulation of sufficient samples. T S H was measured by R I A during routine clinical tests at Cadham provincial Laboratory (Winnipeg, MB) from clotted blood samples of 8 control and 6 mdx mice. CK levels were determined within 3 days of serum collection using a CK-10 creatine kinase diagnostics kit (Sigma Chemical Co., St. Louis, MO) and spectrophotometer (340 nm). Appropriate standards specified by the manufacturer were run in a calibration curve at the time of each assay. Pituitary GH assay Mouse G H was measured in pituitary extracts by r a d i o i m m u n o a s s a y using m o u s e G H s t a n d a r d s (AFP10783B) provided by Dr. A.F. Parlow (HarborU C L A Medical Center, Torrance, CA), and monkey anti-rat G H antisera (NIDDK-anti-rGH-S-5) provided by the National hormone and pituitary program ( N I D D K , Bethesda, MD). Pituitaries were extracted by thorough trituration in 200 #1 of 0.01 M Tris buffer (pH 7.5) containing 0.1% Triton X-100, and centrifugation (1000 × g for 5 rain). Aliquots of supernatant were frozen overnight, and diluted 1:5000 in assay buffer prior to assay. All samples were measured in one assay. Assayed G H concentration was expressed per mg of pituitary tissue. Statistics Data (mean +_ SEM) were compared by Student's t-test or analysis of variance ( A N O V A ) where appropriate. Duncan's multiple range test was used in the latter case to test differences between individual groups when A N O V A was significant. Linear regression on T4 data was run using N W A Statpak (NorthWest Analytical, Inc., Portland, OR). A probability of p < 0.05 was used to reject the null hypothesis.

3. Results

3.1. Thyroid gland weight During dissection, the size of both pituitary and thyroid glands appeared larger in mdx than in agematched control animals. The weight of the thyroid gland from young (2 month old) rndx mice (23.3 +_ 2.7 mg) was significantly greater ( p < 0.001) than thyroid weight from age-matched control mice (14.7 + 1.4 mg). However, the thyroid gland weight relative to body weight in mdx mice was not different from the ratio in

'-;()

)' 0

P' "13

"~w 6.0 v

2/

%7 . -

•

, •

.S: ....... ,

•

,)

"

E / >

1:) (/"

/

,q-

o" E

~77,

':::

L _

4 0

L

•~7 --

- O

O 2 0

10

9 (]

:

~10

£0

60

age (weeks) Fig. 1. Graph (scatter diagram) showing serum T4 levels in 22 control (solid circles) and 31 md~ mice (open triangles), aged between 2 and 56 weeks. Third order regression lines of T4 against age, and 95% confidence limits for each of control (solid lines) and mdx (dashed lines) strains are drawn. There is no difference between the regres: sions, and no significant relationship to age in control mice.

control mice. Pituitary tissue dried too quickly during weighing in tissue required for electron microscopy.

3. 2. TSH, T4 and CK assays T4 data are presented as a scatter diagram in Fig. I. Third order regression lines and their 95% confidence intervals for both control and mdx data, are shown. There was no significant difference in the mean levels of T4 between control and mdx mice (mean *_ SEM: control 4.87 + 0.24 m g / d l : mdx 5.25 _+ 0.15 m g / d l : t(d.f = 5 1 ) = 1.42. p = 0.08). T h e r e was no significant relationship of serum T4 to age m the control strain (r 2 = 0.09. F(3,21) = 0.7). although T4 and age were weakly related in the mdx mice (r 2 = 0.26. F(3.28)= 3.3. p < 0.05). likely due to the small variation of data points in the mdx samples, Blood T S H levels were not significantly different between control (0.028 +_ 0.005. n = 81 and mdx mice (0.027 +_ 0.006, n = 6). Serum C K levels in control animals (56.9 ± 14.5 U/1) were within the normal range of the assay (15-110 U / l ) . In 2-month-old mdx mice. the enzyme level (359.0 z 219.8 U / l ) was greater than in controls ( p < 0.05. d.f = 11), during the characteristic muscle degeneration in young dystrophic animals (Bulfield et al 1984).

,LI:. Anderson et aL /Jourmd ~]'the Neurological Science 123 (1994) 80 87

3.3. Electron microscopy The fine structure of the control mouse pituitary was very similar to that previously reported for rat (Anderson and Thliveris 1979). The typical pituitary cctl typcs were observed, including somatotrophs, gonadotrophs, thyrotrophs, non-granulated cells (Fig. 2a), as well as mammotrophs and corticotrophs. Cells were classified on morphological criteria which included relative cell size and nucleus-to-cytoplasm ratio, and on the location, density, and size of secretory granules within and between cell types (Costoff 1973; Moriarty 1973: Anderson and Thliveris 1979). As the distribution of cell types varies markedly between regions within the pituitary lobes (Baker and Jaffe 1975), their relative numbers were not counted. q'he anterior pituitary of both old and young mdx mice (Fig. 2b) showed similar composition by the six cell types. However, nearly all of the 79 somatotrophs seen in md~" pituitaries were clearly marked by obvious dilation of the rough endoplasmic reticulum (RER) vesicles compared to that in 53 control somatotrophs. Tile golgi apparatus, directly concerned with packaging GH, was enlarged compared to controls, indicating an increase in their activity (Anderson and Thliveris 1979). mdx somatotrophs also contained many dense secretoo' granules of homogeneous size, while control soma-

83

totrophs generally contained fewer granules. Pituitaries from young mdx mice also included some corticotrophs which had enlarged golgi stacks, in addition to the very active somatotrophs. Thyroid glands from 12-month-old control (Fig. 3a) and mdx (Fig. 3b) mice showed the typical cuboidal epithelium around colloid in each follicle. Follicle size seemed larger in mdx than control thyroids, but this was not consistent between animals. Thyroid glands from older animals of both strains also included some atrophic or "cold" follicles, typical of aged mice and involuting follicles in rats (Tachiwaki and Wollman 1990). The ultrastructure of control (Fig. 3c) and rndx (Fig. 3d) thyroid was characteristic of other reports (Tachiwaki et al. 1990; Wollman et al. 1990), and showed the active RER typical of active secretory cells. The RER varied in degree of dilation between cells and between follicles, but was not different between control and mdx thyroids. Granulated calcitoninsecreting cells were also present in the epithelium of both control and mdx tissues. 3.4. GH assay

The weight of pituitaries from age-matched control and mdx mice did not differ significantly, and controls had increased (p _<0.05) gland weight with age (Table

Fig. 2. Electron micrographs of control (a) and mdx (b and c) mouse anterior pituitary glands. (a): In control pituitary, somatotroph cells (S) contain dense granules of homogeneous size within non-dilated cisternae of RER and golgi. Gonadotroph cells (G) contain granules of heterogeneous size. and thyrotrophs (T) contain the smallest secretory granules. (b and c): Electron micrographs of mdx mouse anterior pituitary gland showing hypertrophic somatotrophs (S) containing dilated rough endoplasmic reticulum, prominent golgi regions and a dense nucleus, gonadotroph (G), a thyrotroph (T), and corticotroph (C) with a peripheral array of secretory granules near a large nucleus. X5000 bar = 1 um.

84

J.E. Anderson et al. / Journal of the Neurological Science 123 (1994) 80-87

Fig. 3. Light (a and b) and electron (c and d) micrographs of control (a and c) and mdx (b and d) thyroid gland tissue, a and b: Low magnification views of control (a) and mdx (b) thyroid tissue showing follicles of variable size surrounded by a basement membrane (arrow), which contain colloid deposits (c). Follicles appear to be larger in mdx thyroid gland. Some variation in epithelial thickness (from squamous to cuboidal) is noticeable in both strains. X200 bar = 50 tzm. c and d: Ultrastructure of both control (c) and rndx (d) thyroid shows cuboidal epithelial ceils on a basement membrane (arrow). Thyroidal cells contain a prominent nucleus and rough endoplasmic reticulum ~rer) variably dilated and filled with homogeneous material. Colloid (c) inside part of one follicle, and a granulated calcitonin cell (cc) are also shown. X5000 bar = 1 .tLm.

1). R e l a t i v e G H c o n t e n t was g r e a t e r in o l d e r ( 8 - 1 0 m o n t h - o l d ) m d x f e m a l e m i c e t h a n in a g e - m a t c h e d control f e m a l e s ( p < 0.05, D u n c a n ' s test). N o significant d i f f e r e n c e s d u e to sex ( p = 0.14) or strain ( p = 0.11) w e r e f o u n d by two-way A N O V A (d.f. = 21). O t h e r a g e - b a s e d c o m p a r i s o n s w e r e n o t m a d e d u e to small or a b s e n t groups.

4. Discussion This study o f the p i t u i t a r y - t h y r o i d axis has d e m o n st r at ed that mdx m i c e are e u t h y r o i d t h r o u g h o u t t he d y s t r o p h i c p r o cess (at least up to 56 w e e k s of age), and specifically d u r i n g e l e v a t i o n of s e r u m CK. I n addition. pituitary m o r p h o l o g y indicates no c h a n g e in t h y r o t r o p h

.I.E. Anderson et al. /Journal Of the Neurological Science 123 (1994) 80-87

Table 1 Pituitaa, weight (mg) and growth hormone content (p.g/mg pituitmy) in young (2-month-old) and older (8-10-month-old) male (M) and fiemale (F) control and mdr mice Group Age Sex Pituitaryweight Growthhormone (months5 (mg) n (#g/mg) n ('ontrol

2

C~mlrol S- 10 md.~:

8-1!!

F M F M F M

1,37±0.25 l(5 391 ± 122 1(5 1,13:~0.13 6 1465±585 6 3.22±(5.31 9 ~ 1963±616 9 ' 3.31 I 1188 1 3.22±0.56 8 3021±686 6b 2.01 ±0.65 6 1842 ± 9915 4

l)ata are mean.t standard error (n-sample size). ~' Indicates significant difference from young female controls. b Indicates significant difference from old female controls. activity, but suggests an increased level of synthetic activity (Rennels and Herbert 1980) in somatotrophs. Pituitary G H levels (measured from a different group of mice) confirm the increased synthesis of G H in 8-10-month-old female mdx mice. GH, unlike TSH, is episodically released from the pituitary with the stress of anesthesia (Collu et al. 1970; Martin 1980), so serum G H was not measured. In retrospect, IGF-I levels in serum would have correlated the IGF-1 response of mdx muscle and extractable pituitary G H (Ullman and Oldfors 1989; Murphy et al. 1987). The mdx pituitary-thyroid axis was originally examined for a possible reduction in function which might alleviate m d r dystrophy. This hypofunction has now been ruled out, as T4, TSH, thyrotroph and thyroid follicular cell morphology are normal. Thyroid abnormality has been reported in avian muscular dystrophy (King and Entrikin 1991), and in human myotonic dystrophy (Fukazawa et al. 1990) but not in DMD. However, the findings of m d r somatotroph hypertrophy and high pituitary G H in older m d r females, do indicate some involvement of the endocrine system in rndx mice. Interestingly, the promotion of fiber growth in vivo by high GH, and the muscle fiber hypertrophy in limb muscles of mdx mice with non-lethal dystrophy, appear to be at odds with previous reports that small fibers may be relatively "spared" from dystrophic injury (Karpati et al. 1983, 1988; Moschella and Ontell 1987: Anderson 1991). However, the idea that fiber susceptibility to dystrophic insult results from an interplay of mechanical strain and surface area-to-volume ratio in myofibers (Petrof et al. 1993), possibly in the context of utrophin expression in small D M D and mdx myofibers (Matsumura et al. 1992), helps to account for the discrepancy. We have recently shown that atrophic m d r limb myofibers, produced by anabolic steroid treatment, exhibit increased rather than less dystrophic damage (Krahn and Anderson 1994), so fiber size alone does not strictly determine the severity of dystrophy. In addition, while the present study shows that large fibers, and hypertrophic m d x limb muscles and

85

heart may reflect growth promotion by GH, precursor cell activity is the basis for regenerative capacity. The mechanisms enabling muscle regeneration in the m d x mouse are not completely understood, although the response includes active satellite cell proliferation (Anderson et al. 1987), and myoblast proliferation in vitro (Anderson et al. 1992). Since the increased rndx muscle content of basic fibroblast growth factor (bFGF) (Anderson et al. 1991: DiMario et al. 1989), is clearly localized in newly regenerating m d r myotubes, b F G F likely has some role in proliferation of rndx muscle precursors (Anderson et al. 1991) which may differ from D M D and dog dystrophy, where b F G F is lower (Anderson et al. 1993). Muscle cell commitment is controlled by regulatory genes (reviewed by Weintraub et al. 1991; Wright 1992), and species-specific modifications of precursor cell proliferation during repair are known (Grounds and McGeachie 1989, 1990). As well, growth factors such as b F G F stimulate myoblast mitogenesis (Allen et al. 1984; Gospodarowicz et al. 1987; Baird and Walicke 1989; Florini and Magri 1989) and fetal isoform expression (Parker ct al. 1990) which are also influenced by TH. T H inhibits cardiac myoblast proliferation in vitro (Kardami et al. 1991), and the mitogenic stimulus of b F G F (Clegg et al. 1987) is decreased by TH, since it induces muscle differentiation (Kardami et al. 1988; Kardami 1990; Liu et al. 1993). From the present study, high G H (and likely expression of IGF-I) in a euthyroid milieu, in addition to high b F G F (Anderson et al. 1991, 1993) may optimize muscle precursor proliferation. Since low G H did not alleviate autosomal muscular dystrophy in mice (Coakley et al. 1989), this study suggests that high, rather than low G H supports growth and formation of new muscle in mdx mice. Indeed, the hypertrophy of some fibers and some limb muscles (posterior compartment calf muscles) in human D M D and in dystrophin-deficient cats and dogs (reviewed in Partridge 1991), as well as mdx limb and heart muscle (Anderson et al. 1987, 1988, 1994) suggests that differential muscle and fiber growth may be generally involved in muscle regeneration in X-linked dystrophy. If high G H secretion is only present in mdx mice, examination of G H - d e p e n d e n t gene expression, particularly IGF-I and bFGF, in muscle may reveal new treatments for DMD. The possible promotion of mdx muscle regeneration by G H influences on myobIast proliferation, myotube formation, and fiber growth, in concert with bFGF, IGF-1 and muscle regulatory genes, requires investigation.

Acknowledgements The authors are grateful for the technical assistance of Mrs. D. Love, and Mr. R. Simpson. (}rants from the

8~

J.E. Anderson et al. /Journal qi" the Neurological Science I23 (19941 8 0 - 8 7

Muscular Dystrophy Association of Canada (JEA), Manitoba Medical Service Foundation (JEA), Medical Research Council of Canada (EK) and Manitoba Heart and Stroke Foundation (EK) supported this investigation. EK holds a Medical Research Council Research Scholarship.

References Allen, R.E., Dodson, M.V. and L.S. Luiten (1984) Regulation of skeletal muscle satellite cell proliferation by bovine pituitary fibroblast growth factor. Exp. Cell. Res., 152: 154-160. Anderson, J.E. (1991) Dystrophic changes in rndx muscle regenerating from denervation and devaseularization. Muscle Nerve, 14: 268-279. Anderson, J.E. and J.A. Thliveris (1979) Ultrastructure of the fetal rat anterior pituitary gland at term and during prolonged gestation. Anat. Rec., 194: 257-266,. Anderson, J.E., Ovalle, W:K. and B.H. Bressler (1987) Electron microscopic and autoradiographic characterization of hindlimb muscle regeneration in the rndx mouse. Anat. Rec., 219: 243-257. Anderson, J.E., Bressler, B.H. and W.K. Ovalle (1988) Functional regeneration in the hindlimb skeletal muscle of the mdx mouse. J. Muscle Res. Cell Motil., 9: 499-515. Anderson, J.E., Kao, L., Bressler, B.H. and E. Gruenstein (1990) Analysis of dystrophin in fast and slow twitch skeletal muscles from mdx and dy 2J mice at different ages. Muscle Nerve, 13: 6-11. Anderson, J.E., Liu, L. and E. Kardami (1991) Distinctive patterns of basic fibroblast growth factor (bFGF) distribution in degenerating and regenerating areas of dystrophic (mdx) striated muscles. Dev. Biol., 147: 96-109. Anderson, J., Greenway, S. and E. Scott (1992) Effects of cyclical activity and bFGF on mdx dystrophic and control myoblasts in vitro. Biophys. J., 61 (2, part 2): A452. Anderson, J.E., Kakulas, B.A., Jacobsen, P.F., Johnsen, R.D., Kornegay, J.N. and M.D. Grounds (1993) Comparison of basic fibroblast growth factor in Xqinked dystrohin-deficient myopathies of human, dog and mouse. Growth Factors, 9: 107-121. Anderson, J.E., Liu, L. and E. Kardami (1994) The effects of hyperthyroidism on muscular dystrophy in the mdx mouse: greater dystrophy in cardiac and soleus muscle. Muscle Nerve, 17: 64-73. Baird, A. and P.A. Walicke (1989) Fibroblast growth factors. Br. Med. Bull., 45: 438-452. Baker, B.L. and R.B. Jaffe (1975) The genesis of cell types in the adenohypophysis of the human fetus as observed with immunocytochemistry. Am. J. Anat., 143: 137-162. Bulfield, G., Siller, W.G., Wight, P.A.L. and K.J. Moore (1984) X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc. Natl. Acad. Sci. USA, 81: 1189-1192. Butler-Browne, G.S. and R.G. Whalen (1984) Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle. Dev. Biol., 102: 324-334. Coakley, J.H., Jackson, M.J., Wagenmakers, A.J.M., Ensor, D. and R.H.T. Edwards (1989) Effect of mazindol on dystrophic mice and on growth in young rats. Comp.' Biochem. PhysioL, Part C, 92(2): 385-389. Clegg, C.H., Linkhart, T.A., Olwin, B.B. and S.D. Hauschka (1987) Growth factor control of skeletal muscle differentiation: Commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor. J. Cell Biol., 105: 949-956. Collu, R., Jequier, J.C., Letarte, J., Leboff, G. and J.R. Ducharme (1973) Effect of stress and hypothalamic deafferentation on the secretion of growth hormone in the rat. Neuroendocrinology, 11: 183-190.

Costoff. A. t1973) Hormonal activities and biochemical properties of isolated granule fractions. In: Ultrastructure of Rat Adenohypophysis. Correlation with Function. Academic Press. New York. NY. pp. 180-189. Coulton, G.R.. Curtin. N.A.. Morgan. J.E. and T.A. Partridge ~1988) The mdx mouse skeletal muscle myopathy. II. Contractile properties. Neuropathol. Appl. NeurobioL 14:299-314 D'Albis. A.. Lenfant-Guyot. M.. Janmot. C.. Chanoinc. C.. Weinman. J. and C . L Gallien (1987) Regulation by thyroid hormones of terminal differentiation in the skeletal dorsal muscle. Dev. Biol.. 123: 25--32. DiMario. J.. Buffinger, N.. Yamada, S and R.C. Strohman (1989) Fibroblast growth factor in the extracellular matrix of dystrophic (mdx) mouse muscle. Science. 244: 688-690. Draeger, A.. Weeds. A. and R.B. Fitzsimons (1987) Primary, secondary and tertiary myotubes in developing skeletal muscle: a new approach to the analysis of human myogenesis. J. Neurol. Sci.. 11: 19-43. Florini. J.R. and K.A. Magri 11989) Effects of growth factors on myogenic differentiation. Am. J. Physiol.. 256:C701-(711. Fukazawa. H.. Sakurada. T.. Yoshida. K.. Kaise. N.. Kaise. K.. Nomura. T.. Yamamoto. M.. Saito. S.. Takase. S. and K. Yoshinaga (1990) Thyroid function in patients with myotonic dystrophy. Clin. Endocrinol.. 32: 485-490. Gospodarowicz. D.. Neufeld. G. and L~ Schweigerer (1987) Fibroblast growth factor: structural and biological properties. J. Cell. Physiol.. (Suppl.) 5: 15-26. Grant. A.L.. Helferich. W.G.. Kramer. S.A.. Merkel. R.A. and W.G. Bergen (1991) Administration of growth hormone to pigs alters the relative amount of insulin-like growth factor-I mRNA in liver and skeletal muscle. J. Endocrinot.. 130: 331--338. Grounds. M.D. and J.K. McGeachie (1989) A comparison of muscle precursor replication in crush-injured skeletal muscle of Swiss and Balbc mice. Cell Tissue Res.. 255: 385-391. Grounds. M.D. and J.K. McGeaehie (1990) Myogenic cell replication in minced skeletal muscle isografts of Swiss and Batbc m~ce. Muscle Nerve. 13: 305-313. Guler. H.-P.. Zapf, J.. Scheiwiler. E. and E.R. Froesch (1988) Recombinant human insulin-like growth factor I stimulates growtll and has distinct effects on organ size m hypophysectomized rats. Proc. Natl. Acad. Sci. USA. 85: 4889-4893. Hoffman. E.P.. Brown, R.H. Jr. and L.M. Kunkel (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 51: 919-928. Isgaard. J.. Nilsson. A.. Vikman, K. and O.G.P. lsaksson (1989} Growth hormone regulates the level of insulin-like growth factor-I mRNA in rat skeletal muscle J. Endocrinol.. 120: 107-112. Jennische. E. and G.L. Andersson (1991) Expression of GH receptor mRNA in regenerating skeletal muscle of normal and hypophysectomized rats. An in situ hybridization study. Acta Endocrinol. (Copenh.), 125:595-602 Kardami. E.. Spector. D. and R.C. Strohman (1988,~ Heparin inhibits skeletal muscle growth in vitro. Dev. Biol.. 126: 19-28. Kardami. E.. Liu. L. and B.W. Doble ~1991) Basic fibroblast growth factor in cultured cardiac myocytes. Ann. N.Y. Acad. Sci., 638: 244-255. Karpati, G. and S. Carpenter (1986) Small-caliber skeletal muscle fibers do not suffer deleterious consequences of dystrophic gene expression. Am. J. Med. Genet., 25: 653--658. Karpati, G.. Armani, M., Carpenter, S. and S. Prescott (I983) Reinnervation is followed by necrosis in previously denervated skeletal muscles of dystrophic hamsters. Exp. Neurol., 82: 358-365. Karpati, G., Jacob P., Carpenter, S. and S. Prescott (I985) Hypophysectomy mitigates skeletal muscle fiber damage in hamster dystrophy. Ann. NeuroL, 17: 60-64, Karpati, G., Carpenter, S. and S. Prescott (1988) small-caliber skeletal muscle fibers do not suffer necrosis in mdx mouse dystrophy. Muscle Nerve. 11: 795-803.

,I. E. Ander~on el al. / J o u r m d Of lhe Veurolm,,ical Science 123 (1994) 8 0 - 8 7 King, D.B. and R.K. Entrikin 119911 Thyroidal involvement in the expression of avian muscular dystrophy. Life Sci., 48:9[10 916. Krahn, M.J. and ,I.E. Anderson ( 19941 The effects of anabolic steroid treatment on musctdar dystrophy in the m d r mouse. J. Neurol. Sci, (submitted). lzumo, S., NadaI-Ginard, B. and V, Mahdavi 119861 All members of the M t l C nmltigene family respond to thyroid hormone in a highly tissue-specific manner. Science, 231: 597-600. Izumo. S.. Nadal-Ginard. B. and V. Mahdavi (1990) The thyroid hormone receptor a gene generates functionally different protein isotorms by alternative splicing, In: Roberts, R. and Schneider, M.D. (Eds.), Molecular Biology of the Cardiovascular System, vol. 131, Ahm R. Liss, New York, NY, pp. 111-123. Lathrop, B., Olson, E. and L. Glaser 11985) Control by fibrohlast growth factor of differentiation in the BC~HI muscle cell line, J. ('ell Biol., 100:1540 1547. L,inkhart, T,A.. Clegg, C.H. and S.I). Hauschka 119811 Myogenic differentiation in permanent clonal mouse myoblast cell lines: regulation by macromolecular growth factors in the culture medium, l)ev. Biol.. 86: 19-30. Liu, 1,. and E. Kardami (1991) Hypothyroidism favours expression of high molecular weight basic FGF in the heart. J. Cell. Biochem., (Suppl.) 151': 171. Liu. L,, Doble, B.W. and E. Kardami (1093)Perinatal phenotype and hypothyroidism are associated with elevated levels of 21.5- to 22-kDa basic fibroblast growth factor in cardiac ventricles. Dev. Biol., 157:5117-516. Martin, J.B. (19801 Functions of the central nervous system neurotransmitters in regulation of growth hormone secretion. Fed. Proc., 39: 2902-2906. Matsumara, K., Ervasti, J.M.. Ohlendieck, K., Kahl, S.D. and K.P. Campbell (19921 Association of dystrophin-related protein with dystrophin-assoeiated proteins in mdx mouse muscle. Nature, 3611: 588-591. Mclntosh, L.M., Pernitsky, A.N. and J.E. Anderson 11994) The effects of altered metabolism (hypothyroidism) on muscle repair in the md~ dystrophic mouse. Muscle Nerve, 17: in press, Mortar(y, G.C. 11973)Adenohypophysis: ultrastructural cytochemistry. A review. J. Histochem, Cytochem., 21: 855-894. Moschella, M.C. and M. Ontell 119871 Transient and chronic neonatill denervation of murine muscle: a procedure to modify the phenotypic expression of muscular dystrophy. J. Neurosci., 7: 2145-2152. Murphy, L.J., Bell, G.I., Duckworth, M.L. and H.G. Friesen 11987) Identification, characterization and regulation of a rat complementary deoxiribonucleic acid which encodes insulin-like growth factor-l. Endocrim)logy, 121: 684-691. Nonaka, I., Takagi, A. and H. Sugita (1981) The significance of type 2(" muscle fibers in Duchenne muscular dystrophy. Muscle Nerve, 4:326 333 Nwoye. L., Momaerts, W.F.H.M., Simpson, D.R., Seraydarian, K., and M. Marusich 11982) Evidence for a direct action of thyroid hormone in speciDing muscle properties. Am. J. Physiol., 242: R401 - R408. Orlowski, J. and ,I.B. Lingrel (199111 Thyroid and glucocorticoid hormones regulate the expression of multiple Na,K-ATPase genes in cultures neonatal rat cardiac myocytes. J. Biol. Chem., 265: 3462-3471/. Parker, T.G., Packer, S.E, and M.D. Schneider (1990) Peptide growth factors can provoke "fetal" contractile protein gene expression in rat cardiac myocytes. J. Clin. Invest., 85: 507-514. Partridge, T. 11991 ) Animal models of muscular dystrophy - - what can they teach us? Neuropathol. Appl. Neurobiol., 17:353 363. Petrof, B.J., Shrager, J.B., Stedman, H.H.. Kelly, A.M. and H.L., Sweeney (1993) Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc. Natl. Acad. Sci. USA, 00: 3710-3714. Rennels, E.G. and D.C. Herbert (199(/) Functional correlates of anterior pituitary cytology. Int. Rev. Physiol., 22: 1-40.

87

Salvia(i, (;., Zeviani, M., Betto.. R., Nacamulli, D, and B. Busnardo (I 985) Effects of thyroid hormones on l he biochemical specialization of human muscle fibers, Muscle Nerve, 8: 363-371. Schiaffino, S., Gorza, L., Dones, 1,, Cornelio, F. and S. Sartore (19861 Fetal myosin immunoreactivity in human dystrophic muscle. Muscle Nerve, 9:51 58. Simon ides, W.S. and (7. van Hardeveld (19891 The postnatal development of sareoplasmic reticulum Ca ~ + transport activity in skeletal muscle of the rat is critically dependent on thyroid hormone. Endocrinology, 124:1145-1153. Somerland, H., Ullman, M., Jennische, E., Skottner, A. and A. Oldfl)rs (19891 Muscle regeneration. I h e eflcct of hypophysectomy on cell proliferation and expression of insulin-like growth factor-l. Acta Neuropathol., 78: 264-26LL Taehiwaki, O. and S.ft. Wollman 119901 Comparison of a special class o1 epithelial cells in hyperplastic thyroids undergoing involution and in thyroids in hypophysectomized rats. Am. J. Anat., 189: 57-61. Tachiwaki, O., Zeligs, J.D. and S.H. Wollman 110901 Ultrastructural changes in thyroid epithelium during involution of the hyperplastic thyroid gland. Am. J. Anat.. 189:45 5,5. Totsuka, T., Watanabe, K. and S. Kiyono (19811 Masking of a dystrophic symptom in genotypically dystrophic dwarf mice, I'roc. Jpn. Acad. Sci. B, 57: 1119-113. Ullman, M. and A. Oldfors (1989) Effects o[ growth hormone on skeletal muscle. 1, Studies on normal adult rats. Acta Physiol. Scand., 135:531 537. Ullman, M. and A. Oldflrrs 119911 Skeletal muscle regeneration in young rats is dependent on growth hormonc. J. Neurol, Sci., 106: 67-74, UIIman, M., Alameddine. H., Skottner, A. and A. Oldtk)rs (19891 Effects of growth hormone on skeletal muscle. 11. Studies on regeneration and denervation in adult rats. Acta Physiol. Scand., 135: 537-543. Weintraub. H.. Davis, R., Tapscott, S., Thaycr, M., Krause, M., Benezra. R., Blackwell, T.K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y. and A.B, Lassar (1991) The MyoD gene family: nodal point during specification of the muscle cell lineage. Science, 251: 76.1-765. Whalen, R.G.. Toutant, M., Butler-Browne, (LS. and S.C. Watkins 11985) Hereditapy pituitary, dwarfism in mice affects skeletal and cardiac myosin isozyme transitions differently. J. Cell Biol.. 101: 603-609. Wollman, S.H., Herveg, J.P. and O. Tachiwaki (19901 Histoh)gie changes in tissue components of the hyperplastic thyroid gland during its involution in the rat. Am. ,1, Anat., 189: 35-44. Wright, W.E. (19921 Muscle basic helix-loop helix proteins and the regulation of myogenesis. Curt. Opinion Gen. Dcv., 2: 243--248. Zaccaria, M., Angelini, C., Dal Pont, O., Pcgoraro, E., Marchetti, M. and C. Scandellari (1989) Duchenne muscular dystrophy: lack of response to arginine test and h)w somatomedin-C levels. In: J.R. Bierich, E. Cacciari and S. Raiti (Eds.), Growth Abnormalities, vol. 56, Serono Symposia Publications, Raven Press, New York, NY, pp. 469-473, Zacharias, J.M. and J.E. Anderson (1901t Muscle regeneration after imposed injury, is better in younger than oMer md.r dystrophic mice. J. Neuroi. Sci., 1114: 190-196. Zatz, M_ Betti, R.T.B. and J.A. Le,,T 119811 Benign Duchenne muscular dystrophy in a patient with growth hormone deficiency. Am. J. Med. Genet., 1(1: 31/1-3(t4. Zatz, M. and O. Frota-Pessoa 119811 Suggestion for a possible mitigating treatment of Duchenne musctdar dystrophy. Am. J. Med. Genet.. 1{1: 304-31/7. Zatz, M., Bet(i, R.T.B, and O. Frota-Pessoa (19861 Treatment of Duchenne muscular dystrophy with growth hormone inhibitors. Am. J. Med, Genet.. 24:549 566.