EXPERIMENTAL NEUROLOGY 57, 47545 (1977)

Elevated Cholesterol in Tissues of Chicken Embryos with Hereditary Myotonic Muscular Dystrophy

PATRICIA A. STEWART, EVA S. WERSTIUK, JOHN D. VICKERS,

AND MICHEL P. RATHBONE 1

Medical Research Conncil of Carlada Grozlp in Developmental Neurobiology, Departmwt of Nezlroscicnces, McMaster University, Hamiltotk, Ontario,

Canada, L8S 4J9

Received Febrnary 15, 1977

The cholesterol content of muscle, liver, serum, and brain was examined both in embryos of chickens with hereditary muscular dystrophy and in normal control embryos between 9 and 16 days in ovo. The superficial pectoral muscles, which are severely affected by muscular dystrophy later in development, have a significantly higher cholesterol content than those of normal embryos at all ages examined. In contrast, the cholesterol content of thigh muscles that are not severely affected by the dystrophic process is no different from that of control embryos. The cholesterol contents of liver and serum from dystrophic embryos are also increased, but the cho- lesterol content of the brain is not. The increased cholesterol contents of liver and serum are due to proportionate increases of both free cholesterol and cholesteryl esters. In contrast, in superficial pectoral muscles the ratio of cholesteryl esters to free cholesterol is significantly increased. These data indicate that, in addition to the elevated cholesterol in the liver and serum of dystrophic embryos, the regulation of cholesteryl ester content in the dystrophic pectoral muscles is abnormal.

IKTKODUCTION

Although the most significant clinical features of hereditary muscular dystrophies are seen in muscles, abnormalities have been described in such diverse extramuscular tissues as red blood cell membranes (6, 7, 35, 36, 39), nerve tissue (1, 14, 18, 20, 23, 32, 3S), liver (33, 39, 40), and ecto- dermal derivatives (8). The activities of membrane-bound enzymes such as the (Nat + K’) iVg?+ ATPase, adenylate cyclase, acetylcholinesterase, pro-

1 Drs. Stewart and Vickers are Postdoctoral Fellows of the Muscular Dystrophy Association of Canada. Dr. Stewart’s present address is Department of Anatomy, University of Toronto, Toronto, Ontario, Canada.

Copyright All rights 9 1977 by Academic Press. Inc.

reproduction in any form resmwl,

475

ISSN 0014-4886

476 STEWART ET AL.

tein kinase, and microsomal calcium transport in muscle, liver, and erythro- cytes are altered in a number of dystrophies (3, 22, 31, 33-36).

The lipid composition of membranes was also reported to be abnormal in erythrocytes of patients with muscular dystrophy (19), in muscles from dystrophic mice (30), and in isolated, fragmented sarcoplasmic reticulum of muscles from adult dystrophic chickens (17). Vallyathan (40) reported that the in vitro rate of oxidation and incorporation of glucose into lipids of muscle and liver of dystrophic chickens was higher than that of normal chickens between 1 week and 12 months after hatching.

In myotonic dystrophy in man, the fluidity of erythrocyte membranes is abnormal (6, 7), and in chicken myotonic dystrophy the fluidity of muscle, liver, and erythrocyte membranes is abnormal (39).

Taken together, those findings have led to the suggestion (39) that myotonic muscular dystrophy in chickens and some human muscular dystrophies may result from a disorder of lipid metabolism that alters the lipid composition of cell membranes. In turn, this could account for the alternations observed in both membrane fluidity and the activities of mem- brane-bound enzymes ( 15).

If changes in the lipid composition of membranes were the primary alteration in muscular dystrophy in chickens, then an altered lipid metabo- lism would be seen as the first event in the course of the disease. Since cholesterol content is a principal factor in determining the fluidity of membranes (27), we have examined whether or not there is an alteration in the cholesterol content of tissues in dystrophic embryos early in develop- ment.

MATERIALS AND METHODS

Dystrophic Clzickens

Eggs from chickens homozygous for muscular dystrophy were obtained from Dr. L. Pierro, Department of Animal Genetics, University of Con- necticut, Storrs, Connecticut. The dystrophic mutant gene (am) was first detected in a line of New Hampshire chickens (2). The line maintained by Dr. Pierro is derived from these, but has been outcrossed with White Leghorn to improve the viability of the strain. In these animals, the disease is observed clinically 2 to 3 weeks after hatching and is characterized by a progressive inability of the birds to right themselves when placed on their backs. Muscle degeneration is restricted to white fibers and is ap- parent 1 week c.c 02’0 in the superficial pectoral muscles, which are com- posed predominantly of white fibers (10, 11).

Eggs from White Leghorn chickens were obtained from the Department of Animal Husbandry, University of Guelph, Guelph, Ontario, and from Martindale Hatchery, R.R. 3, Caledonia, Ontario. The eggs were stored at 15°C until they were incubated in a forced-draft incubator at 39°C at a relative humidity of 50 to 60%.

Cholcstcvol Dctcrrlrhations

(u) Preparation of S~nlm. Approximately 100 ~1 whole blood was re- moved from a large vein in the chorioallantoic circulation of embryos at 9 to 16 days in oz’o. The blood was allowed to clot 2 h at room temperature. After centrifugation 2.5 min at 8000 g, 50 ~1 serum were removed for determination of cholesteral content.

(b) Preparation of Tissues. Muscle, liver, and brain were dissected from embryos under a dissecting microscope. The tissue was placed in a plastic microtube which was sealed immediately to prevent weight loss by evaporation. Tissue weight was determined as the difference between the weight of the tube containing the tissue and the weight of the empty tube after tissue was removed. At 9 days iw 0~10 it was necessary to pool both superficial pectoral muscles from each of two embryos to obtain enough tissue for cholesterol determinations. At 10 and 11 days ilz 02’0, both superficial pectoral muscles from a single embryo were sufficient. In all older embryos, either the left or the right superficial pectoral muscle was analyzed.

(c) Cholcstcrol E.t-tracfion. Fifty microliters serum were added to 500 ~1 2-propanol. This was mixed continuously on a mechanical shaker 10 min to ensure complete extraction of cholesterol. Precipitated protein was removed by centrifugation for 2.5 min at 8000 g. Duplicate samples of 100 ~1 supernate were removed for determination of cholesterol. Tissue Sam- ples weighing 20 to 50 mg were homogenized in 500 ~1 3-propanol using ground-glass homogenizers (Micrometric Instrument Co., Cleveland, Ohio). The homogenate was transferred to plastic tubes which were then sealed and shaken 10 min on a mechanical shaker. The denatured protein was removed by centrifugation for 2.5 min at SOOOg. Duplicate samples of 100 ~1 supernate were removed for determination of cholesterol. Com- pleteness of extraction of muscle cholesterol by 2-propanol was confirmed by comparing the amount of cholesterol extracted by that technique with the amount extracted using the method of Folch et al. (16) as modified by Organisciak and Klingman (29). The extraction procedures removed similar amounts of cholesterol. Therefore, the simpler 2-propanol extrac- tion technique was used routinely in the subsequent experiments.

418 STEWART ET AL.

(d) Cholesterol Assay. Cholesterol was assayed by a modification of the method of Leffler and McDougald (21). One milliliter 0.1% (w/v) FeC& in glacial acetic acid was added to 500 pl 2-propanol that contained the sample of cholesterol to be assayed. After mixing, the samples were set aside 10 min at room temperature. One milliliter concentrated sulfuric acid was layered under the solution, and the whole mixture was mixed immediately and thoroughly using a Vortex mixer, The optical density of this mixture was determined at 540 nm after 10 min. Unknown sample concentrations were determined by comparison with standard samples that contained 0, 10, 20, 30, 40, and 50 pg cholesterol standard (Sigma Chemical Co., St. Louis, Missouri) in 500 ~1 2-propanol. Heating the re- action mixture (25) had no effect on the final extent or stability of the color that developed. Apparently, sufficient heat was produced during the reaction with sulfuric acid to ensure maximal color development.

(e) Determination of Free and Esterijied Cholesterol. Free and esterified cholesterol were separated by thin-layer chromatography on 250~pm Anasil H plates (Analab, North Haven, Connecticut) using petroleum ether (30 to 6O”C)-ether (80: 20, v/v) as the developing solvent as described by Beukers et al. (4). Tissue samples were homogenized in 500 $ 2- propanol, as above. After mixing and centrifugation, duplicate 200-~1 portions were removed and dried under a stream of dry Nz, and the residue was dissolved in 50 ~1 methylene chloride-methanol mixture (2: 1, v/v) and transferred quantitatively to the thin-layer plates. The chromato- grams were developed 30 min and allowed to dry. The sports that con- tained free and esterified cholesterol were visualized with iodine vapor and their positions were marked. The appropriate areas (free cholesterol : RF = 0.13 ; esterified cholesterol : R, = 0.82) were removed, using the Ana-Vat sample remover (Analab, North Haven, Connecticut) and the samples were eluted with a mixture of methylene chloride-methanol (2 : 1, v/v, 3 x 1 ml) directly into the calorimeter tubes. The recovery of the free cholesterol was better than 80%, and that of cholesteryl oleate was better than 90%. The organic solvent was evaporated under dry Nz, and the amounts of free and esterified cholesterol were determined using o- phthalaldehyde (Nutritional Biochemicals Corp., Cleveland, Ohio) as de- scribed by Rude1 and Morris (37). Concentrations in unknown samples were determined from standard curves obtained using 10 to 90 nmol of either standard cholesterol or cholesteryl oleate solutions (Analab) .

RESULTS

Total Cholesterol Content of Normal and Dystrophic Tissues

The cholesterol content of the superficial pectoral muscles of normal embryos increased from 5.54 k 0.16 nmol/mg wet weight (mean value

CHOLESTEROL CHOLESTEROL nmoledmg wel wt nmoled~l

12 _

PECTORAL MUSCLE SERUM

II _

,LI I I I I I I 1 L, I 1 I 1 1 I 9 10 11 12 14 16 9 10 11 12 14 16

DAYS IN OVO

FIG. 1. Mean cholesterol content (?SE) of superficial pectoral muscles and serum

from normal (0-O) and dystrophic ( l - - - - -0) chicken embryos from 9 to 16

days i~z OVU. The number of experiments at each stage of development is indicated in parentheses.

* SE) at 9 days in azlo to 7.00 -+ 0.47 nmol/mg wet weight at 16 days i~t ozlo (Fig. 1A). There was no significant change in the cholesterol con- tent of normal pectoral muscles between 10 and 12 days in OZQ. The cholesterol content of the superficial pectoral muscles from dystrophic embryos also increase between 9 and 16 days in OZJO from 6.31 t 0.08 to 10.09 k 0.23 nmoles/mg wet weight. However, the cholesterol content of the muscles of dystrophic embryos was always significantly higher (0.001 < P < 0.02 among the cases examined) than that of the muscles of normal embryos at each age examined. There was no dii-ference (P > 0.2) in cholesterol content between the right and left pectoral muscles from single embryos at 12 to 16 days ilz oz’o. It was not possible to analyze superficial pectoral muscles from embryos younger than 9 days in ozm because the superficial pectoral muscles could not be distinguished from the deep pectoral muscles and surrounding connective tissue.

The cholesterol content of the serum of both normal and dystrophic embryos did not increase significantly until after 14 days ilz oz’o (Fig. 1B). The serum cholesterol of dystrophic embryos was always significantly higher than that of age-matched normal embryos (0.001 < P < 0.01). We also determined the cholesterol content of other tissues at 11 days in oz’o, because during this period there is little change in the value of

480 STEWART ET AL.

TABLE 1

Cholesterol Content of Tissues from Normal and Dystrophic

Chicken Embryos at 11 Days in Ooo

Tissue Number of Normalsa Number of Dystrophies’ P experiments experiments

Serum 17 5.08 f 0.23 13 6.70 f 0.54 <O.OOl

Pectorals 15 6.03 f 0.23 14 7.17 f 0.16 <O.Ol Liver 19 14.28 f 0.52 17 17.49 f 0.62 <O.OOl

Thigh 21 8.46 f 0.28 21 8.56 f 0.34 NS* Brain 12 11.20 f 0.13 12 11.82 f 0.10 NSb

D Nanomoles per milligram tissue or nanomoles per microhter serum; mean values

f SE. * Not significant at the P = 0.05 level of significance.

cholesterol in pectoral muscles or serum. The cholesterol content of livers from dystrophic animals (17.49 5 0.62 nmol/mg wet weight) was sig- nificantly higher than that from normal embryos (14.28 -+ 0.52 nmol/mg wet weight ; Table 1). On the other hand, no differences were observed in the cholesterol content of brain or thigh muscles of normal or dystrophic embryos (Table 1). In these experiments we would have detected differ- ences in cholesterol content of 4.3% or more at the P = 0.05 level of significance.

Free and Esterified Cholesterol in Dystrophic and Normal Tissue

To determine whether or not the observed increase in the cholesterol content of dystrophic embryos was due to elevated free (unesterified) cholesterol or cholesteryl esters, we separated those two fractions by thin- layer chromatography and determined their respective concentrations. Table 2 demonstrates that the amount of cholesteryl esters in superficial pectoral muscles, serum, and livers of dystrophic embryos is increased compared to that of controls. In contrast, the free cholesterol values are increased only in the serum of dystrophic embryos. There was a significant increase in the ratio of esterified to free cholesterol in dystrophic (0.36) compared to normal (0.21) pectoral muscles (N = 6, P < 0.05). In other tissues, the ratio of esterified to free cholesterol was not different in dystrophic and normal embryos.

DISCUSSION

The cholesterol content of the superficial pectoral muscles, serum, and liver is elevated in dystrophic chick embryos as early as 9 days in ovo. This is the earliest biochemical difference between dystrophic and normal

CHOLESTI~KOL IN DYSTROI’IIIC CIIICKEiX EM t3RYOS 481

chickens yet reported; it is present at an early stage of ontogeny, before other features of the disease develop. This finding supports the suggestion that a defect in lipid metabolism may underlie this disorder (17, 2S, 33). The observed elevation in cholesterol content of dystrophic superficial pectoral muscles may be an early manifestation of the lipid accumulation observed histologically in the superficial pectoral muscles of older chickens with muscular dystrophy (12). The elevation of both hepatic and serum cholesterol in the dystrophic embryos is consistent with the major role of the liver in cholesterol metabolism and in regulation of serum cholesterol concentrations (13).

In contrast to the superficial pectoral muscles, the cholesterol content of the thigh muscles was no different in normal and dystrophic embryos. This may be due to the pattern of distribution of the muscles affected by dystrophy in these animals. In chickens, the dystrophic process predomi- nantly affects white fast-twitch muscle fibers. The superficial pectoral muscles, which are composed predominantly of this type of fiber, show clear histologic features of the disease by 1 week ex OZ’O (12). In contrast, the thigh muscles contain a mixture of fast, intermediate, and slow fibers that do not show such marked degeneration. Consequently, if the elevated cholesterol content is associated with the fibers that undergo dystrophic degeneration, less extensive changes in cholesterol content would be ex- pected in thigh muscles than in superficial pectoral muscles.

We have previously demonstrated that the neural tube of dystrophic chicken embryos plays an important role in the pathogenesis of muscular dystrophy in chickens (14, 32). This effect is exerted between 3 and 3.5 days in ozlo. This raises the possibility that either (a) the cholesterol content of dpstrophic and normal nerve tissue is the same, (b) any changes present at 2 to 3.5 clays are lost by 11 days, or (c) any differences

TABLE 2

Free and Esterified Cholesterol Content of Tissues from Normal and

Dystrophic Chicken Embryos at 11 Days in 0~~~

Tissue

Free cholesterols

Normal D,-S- Standard troyhic error of

the mean difference

Esterified cholesterolb P Ko1mal Dys- Standard P

tropllic error of the mean difference

Serum 1.28 1.9R f0.26.3 <a.05 0.86 2.17 *0.355 <(I.02 Pectoral 2.44 2.55 5~0.254 NS’ 0.5 1 0.91 fll.105 <(I.02 Liver 3.15 2.81 zkn.212 NS 1.18 3.29 f0.332 <o.ot Thigh 2.82 2.52 f0.186 i-C.5 0.81 1.16 fO.533 NS Brain 4.86 4.19 zto.490 NS 0.21 0.28 f0.052 KS

-Values are means of 8i.x experiments. ) Expressed as nanomoles per milligram wet weight of tissue or nanomoles per microliters serum. 5 Not significant at the P = 0.05 level of significance.

482 STEWAKT ET AL.

between the cholesterol content of dystrophic and normal nerve tissue may be masked by the high cholesterol content of myelin in the brain (24).

The elevated total cholesterol content of dystrophic livers and serum was due to an increased amount of both cholesteryl esters and free cho- lesterol. Because free cholesterol readily diffuses into plasma membranes (9, 26), the free cholesterol content of the plasma membranes would in- crease. The cholesterol content of membranes is a major determinant of their fluidity (27) and, therefore, the increased free cholesterol in the serum could account for the increase in microviscosity of cell plasma mem- branes reported for dystrophic chickens (39).

The activities of a number of membrane-associated enzymes including (Na’ + K+)Mg”+ ATPase, Ca 2+ ATPase, acetylcholinesterase ( 15)) and endogenous membrane protein kinase (41) are altered by changes in mem- brane cholesterol content. It is possible that cholesterol mediated changes in membrane fluidity may account for the changes in the activities of these membrane-associated enzymes. The activities of these enzymes have been reported to be altered in certain muscular dystrophies (31, 33, 3436, 39). Therefore, caution must be exercised in interpreting the significance of alterations in the activities of membrane-associated enzymes in muscular dystrophy of chickens, as these alterations may be secondary to the increased concentration of free cholesterol in both the plasma and the plasma mem- branes of cells.

In these experiments the total cholesterol content of superficial pectoral muscles was higher in dystrophic than in normal embryos. However, in contrast to all other tissues examined, there was an increase in the ratio of cholesteryl esters to free cholesterol in superficial pectoral muscles of dystrophic embryos. In extrahepatic tissues, normal intracellular cho- lesterol concentrations are maintained by regulating both the rate of up- take of cholesterol from the plasma into the cells and the rate of intra- cellular cholesterol synthesis and metabolism (5). Thus, it appears that the increased cholesterol content of dystrophic pectoral muscles is likely due to their failure to regulate the uptake or metabolism of cholesterol within the cells, rather than resulting directly from the elevated serum cholesterol. Certainly, humans and lower animals that are hypercholesterol- emit do not normally develop muscular dystrophy.

Because cholesterol concentrations are elevated very early in these embryos, long before there is histological evidence of the muscle degenera- tion that has been observed in the disease (12), it appears that the increase in cholesterol may be closely related biochemically to the expression of the dystrophic gene in these tissues, and not a consequence of muscle degeneration.

CHOLESTEROL 1N DYSTROPHIC CHICKEN EMBRYOS 483

ACKNOWLEDGMENT

The authors gratefully acknowledge the technical assistance of Mrs. J. Ainsworth.

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