skeletal muscle lipids in normal and dystrophic ... - Wiley Online Library

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dcscrihcdby Folch, Lees and Sloane Stanley (W57). ..... Department of PalholoL>, King Edward Memorial Hospital for Women, for the use ofthe gas chromal-.
.\JEBAK 58 (Pt. 4) 397-408 (1980)

©SKELETAL MUSCLE LIPIDS IN NORMAL AND DYSTROPHIC MICE by P. H. PEARCE AND B. A. KAKUEAS (Erom Ihc Department of Neuropathology. Royal Perth Hospital, Western Australia, and Department of Pathology, University ofWestern Australia.) (Acceplcil for ptthlicalioti .fuiic 6, 1980.)

Summary. The lipids oF muscle From normal and dystrophic mice of the Bar Harbor strain C57BL/6J uere investigated with particular reFerence to Ihc Fatty acid composition ofthe neutral lipids and phospholipids. Triglyceride, which together wilh cholesterol comprised most oFthe neutral lipid fraction, showed no signiFicant changes in Fatty acid composition. There was no significant change in lhe amount oF lotal phospholipid phosphorus in dystrophic muscle, hut analyses o l t h c levels oF individual phospholipids showed a decrease oF y'*/ii in phosphatid\lcholine. In both phosphalidvlcholinc and ethanolamine phosphoglvceridc. large decreases were observed in the proportion of the Falty acid I6;O, while I 8:0 and 18:1 increased significantly. In phosphatldylcholine 18:2 also increased, and a decrease occurred in 22:6 in cthanolaminc phosphoglyceride. Sphingomyelin showed increases in 16:0, 23:0 and 24:1 and decreases in 18:0 and 18:1. The other phospholipid Fractions showed less remarkable changes, with a significant increase in 18:0 in lysophosphatidylcholine and a decrease in 16:0 in eardiolipin.

INTRODUCTION The Dyslrophia muscularis gene (dy) in mice causes a progressive myopathy whieh exhibits similar histological and pathologieal characteristics in the strains 129/ReJ and C57BE/6J and in the F, hybrid of these strains, 129B6E, (MacPike and Meier., 1976). There is an increase in connective tissue in the dystrophic muscle and fatty infiltration occurs (West and Murphy, I960). Biochemical studies of dystrophic muscle in strain 129 (Shull and Alfm-Slater, 1958; Young, Young and Edelman, 1959; Susheeia, Hudgson and Walton. 1968) and 129B6E, (Owens and Hughes, 1970) have shown increases in total lipid and in the neutral lipidclasses, triglyceride. free fatty acids and cholesterol. There is no change in the total amount of phospholipid (Hughes. 1965; Young ei ai. 1959). although changes have been reported for the phospholipid composition of whole muscle and microsomes of l29B6Ei (Owens and Hughes, 1970) and of sarcolemma of 129/ReJ (De Kretser and Eivett. 1977). Owens and Hughes (1970) also observed marked alterations in the fatty acid composition ofthe total phospholipid fraction from whole muscle and subcellular fractions of dystrophic muscle.

398

P. H. PEARCE AND B. A. KAKULAS

Attention has been focused on the phospholipids because of their role in determining the structure and properties of membranes. Abnormal properties of both sarcolemma and sarcoplasmic reticulum have been reported in muscular dystrophy in strains 129 (Burr and MeEennan, 1961: Martonosi, 1968; Bray, 1973; Boegman. 1974) and C57BE (Uchino, 1977), and it is possible that these differences may be related to the composition ofthe phospholipid fraction and to lhe fatty acid content ofthe individual phospholipids. However, the phospholipids of C57BE have not been investigated; nor has there been a thorough analysis of the fatty acid compositions of the individual phospholipids in muscular dystrophy in any ofthe strains, the data presented by Owens (1968) at a conference being only of a very preliminary nature and quite inadequate for sound comparisons and conclusions. In this study we have examined the lipid composition of muscle from normal and dystrophic mice ofthe strain C57BE/6J, with particular attention being paid to the phospholipids. Significant alterations were found in the dystrophic muscle, especially in phosphatidylcholine, ethanoiamine phosphoglyceride and sphingomyeiin. It is important to remember that, in contrast to muscular dystrophy in humans, murine muscular dystrophy is associated with marked abnormalities of the peripheral nerves (Bradley and Jenkison, 1973). In view of this ditTerence. and of the controversy in the literature about phospholipids in human muscular dystrophy (Plishker and Appel, 1980, for review), we have restricted our considerations to the condition in mice.

MATERIALS AND METHODS Dystrophic mice ofthe Bar Harbor strain C"57BL'6.l-^/y were used, with phenotypically normal litter mutes as controls. The mice were Fed standard mouse pellets and had access to food and water aJ libitum. Animals aged 4-8 months were lightly anaesthetised with ether and killed by cervical dislocation. Muscle from the hind legs was dissected out. freed From visible nerve, fat and connective tissue, frozen in liquid nitrogen and stored at - 7 0 ° . /{islcloiiii ill .studies A small piece oF muscle was fixed in \0% neutral buffered Formalin for 48 h, dehydrated and embedded in paralTm wax. Sections (5 fivn) were cut and stained with haematox>iin and eosin. E.\tnicti(niot lipids The extraction procedure was similar to that of Owens and Hughes (1970) hased on the method dcscrihcdby Folch, Lees and Sloane Stanley (W57). Eaeh analysis included muscle from I normal mouse, or pooled muscle From 2 dystrophic mice. In all. Four analyses were made, i.e. Four normal and four dystrophie. In each case the musele (1 O-t 3 g) was chopped Finely and added to 20 mIchloroForm-methanol (2:1, v/v) containing the anlioxidant BHT (2.6-di-fcr/-butyl-/i-cresoL 5mg'100ml). The tissue was homogenised with an homogeniser with stainless steel blades (Silverson Machines Ltd.. Bucks.) For Four periods each oF30 sec, then allowed to stand For 20 min. After filtration through a sintered glass funnel, the tissue was re-extracted by lhe same procedure. The combined filtrates were evaporated lo dryness at 25° under reduced pressure and the residue was twice redissolved in 8 ml chloroform-methanol-water (64:32:4. v/v/v) and evaporated lo dryness to break protco-lipid bonds. The residue was then dissolved in 10 ml cbloroform-methanol

MUSCLE LIPIDS IN DYSTROPHIC MICE

399

(2:1. v/v) and washed with 2 ml water. The surface of ihc lower phase was rinsed wilh Folch Upper Phase and llie lower phase was evaporated under a stream of nitrogen. Silicic

acidchroniatcyraphv

Silicic acid (BDH Chemicals Lid,. Poole. England) was activated at 150° for 16 h prior to use. A slurry of 4 g silicic acid in chloroform was poured into a column and washed with 1 5 ml chloroform. The lipid extract dissolved in 5 ml chloroform was applied lo the column and the neutral lipid iVaction was eluted under a slighl positive pressure of nitrogen with 100 ml chloroform into a llask containing 4 mg BUT. Pigment, which otherwise sometimes interfered with subsequent thin layer chromatography ofthephospholipids. was eluted with 200 m! acetone and then phospholipids were eluted with 100 ml methanoi into a flask eontaining4 mg BHT, The etuates were concentrated under reduced pressure and stored under nitrogen at - 1 5 ' . fhin kiyenhroDi

All ihin layer chromatography plates were pre-washed h\ running overnight in chloroformniethanol(2:l. v/\)and were activated at 105 for30 min betbre use. Samples to be chromatographed were streaked on the plates and standards were spotted. Alter chromatography. lipids were detected by spraying standards and Ihe outer edges oflhe streaked material wilh iodine dissolved in methanoi (1%. w/v). The lipids were scraped otT under suction into small sintered glass funnels and eluted with appropriate soKents. Uil Nctiirat lipids Neutral lipids were separated on silica gel G plates, thickness 0-5 mm (Prekotes. Applied Science Labs, Inc. Pa.) with petroleum ether (b.p. 60-80°t-dielhyl ether-acetic acid (85:15:1, v/v/v) as solvent. The separated fractions iriglyceride. cholesteryl esters, free fatty acids and diglycerides. were identified from co-chromatographed standards (Sigma Chemical Co. St. Louis. Mo.) and eluted with 10 ml chloroform-methanol (2:1. v/v) into tubes containing 40 /;g BHT, The eluates were evaporated under a stream ol'nitrogen, Ih; Phosphotipids Phospholipids were separaied by one-dimensional thin layer chromatography on silica gel G plates, thickness 0 5 mm. with chlorofornvmelhanoi-acetic acid-water (65:4.1:1:3) (Owens. 1966) as solvent. The separated fractions, pho^phatidylcholine. ethanolamine phosphoglyceride. sphingomyelin. phosphalidylserine + phosphatidyiinositol and lysopliosphalidylcholine. were identified hy comparison wilh co-chromatographed standards (Sigma Chemical Co, and ICN. K & K Labs, Inc, Plain View. N.Y.) and eluted with 3 ml chloroform-metlianol (2:1. v/v). 3 ml chloroform-methanol (1:2. v/v) and 4 ml methanoi into tubes containing 40/jg BHT. Preparation of methyl cslei\ und itinu-thylacctals Methyl esters and dimeth>lacetals were prepared from the separated neutral lipid and phospholipid classes using 14% BF, in methanoi (BDH Chemicals Ltd.) (Morrison and Smith. 1964) in tubes Hushed with nitrogen and sealed with Tellon-lined stoppers. The fatty acid methyl esters and dimethylacetals were e,\tracted wilh hexane. concentrated under a stream of nitrogen and then purified by thin layer chromatography on silica gel 60 (Merck. Darmstadt. W. Germany) wilh petroleum etherdiethylether-acctic acid (85:15:1, v/y/v) as solvent. The purified fraction was eiuted with chloroform (containing 2 mg BHT/100 ml for phospholipid fractions). The eluates were evaporated under a stream ofnitrogen and stored at - 15° under nitrogen until analysed by gas chromatography. Gas

ihromalography

Gas chromatography was carried oul using a Varian .Aerograph 2700 Chromatograph litted with a name ionisation detector. The glass column (I -8 m x 1 -8 mm i,d.) was packed with 10% EGSS-X on Chromosorb W HP. 80/100 mesh (Applied Science Labs,), Helium was used as the carrier gas at a flow rate of 30 nil/min. The temperature programme was from 150° to 2 13° at a rateof 2°/min and then the temperature was held at 21 3° for 10 min. Fatty acid methyl esters were identified by isothermal operation of ihe column al 160° and comparison of retention times with those of standard methyl esters (Sigma Chemical Co. and Applied

P. H. PEARCE AND B. A. KAKULAS

400

Science Labs.). Further identification of equivocal peaks (eg 20:4 and 22:1) was aided by chromatography on the non-polar liquid phase 3% SE-30 (Supeico) on C'hromosorb W 80/100 mesh in a lemperature programme from 130° to 350°at4°/min, Areas ofthe peaks were calculated by triangulalion. Data are presented for those fatty acids which comprised more than I'^o ofthe total. Protein The residue remaining after filtration of ihe initial extracts was incubated overnight at 23" in 0 05 M NaOH (Lilienthal et cd.. 1950), The solubilised non-collagen protein ofthe filtered solution was determined by ihe method of Lowry el cd. (1951) with bovine serutn albumin. Fraction V (Commonwealth Serum Labs.. Melbourne,,Aust,) as standard. Phtisphoriis Phospholipid phosphorus was estimated as described by Owens and Hughes (1470). Sotvcnis Ail solvents were of AR grade and were purchased from BDH Chemicals (Aust,) Pty, Ltd.. V i c or from Aja,\ Chemicals. Sydney. Slali.slicalanalyse.'i Statistical analyses were carried out using S t u d e n t ' s l-test (Snedecor and C o c h r a n , I97X).

RESULTS The results presented in Table I show that there was no significant difTerence between normal and dystrophic muscle with respect to the total amount of lipid, non-collagen protein and phospholipid phosphorus. Examination ofthe tissue microscopically (Fig. IA & B) indicated only a small amount of inftltration ofthe muscle with connective tissue and fat. For example, visual assessment of the normal tissue indicated about 95% muscle and 5% connective tissue with almost no fat, while in the dystrophic samples there was evidence of muscle breakdown with 5-10% connective tissue and 5-10% fat. Neutral lipid.s

The neutral lipid fraction was treated to form trimethylsilyl derivatives (Kuksis, Stachnyk and Holub, 1969), and gas chromatography was performed on a short glass column (0-6 mx2 mm i.d.)of3% Dexsil400on Supelcoport 100, with a temperature programme from 100° to 350° at 47min. The expected pattern was TABLE 1 Total lipid. non-cottagen protein und plwsphotipid phasphoruMifnarmul l Total lipid

Normal Dystrophic

iDul dy.'^iropliic tnousc Phospholipid phosphorus

mg/g wet wt

Non-collagen protein mg/g wet wt

65 6 ± 6.9 810 ± 18.3

157 ± 15 I 34 ± 8

485 ± 22 547 ± 23

wet wt

Total lipid was delermined by weighing the lipid obtained from the initial chlorotbrni-methanol (2:1. v/v) extracts. Values are expressed as means ± S,E,M, for 4 analyses, (Each analysis represents muscle from 1 normal mouse or pooled muscle from 2 dystrophic mice).

MUSCLE LIPIDS IN DYSTROPHIC MICE

401

Fig. 1. A. Normal skeletal muscle in longitudinal section rrom conlrol mouse, hi. & E. x 200 (The scale marker is 100 /mi). B. Longitudinal section of skeletal muscle from dystrophic mouse showing myopathic features. There is disorganization of overall architecture with variation in size of muscle fibres, clumped and crowded nuclei and necrosis with myophagia. Slight increase in connective tissue is also present. H,&E. x 200 (100/jm scale marker).

402

P. H. PEARCE AM) B. A. KAKULAS

obtained (Kuksis. Marai and Gornall. 1967) with major peaks representing triglycerides and cholesterol. There were no marked differences between normal and dystrophic extracts except for an increase in cholesterol as has already been reported (Young cl al, 1959; Owens and Hughes, 1970). No unusual peaks were detected in the profiles from dystrophic mice. When the neutral lipid fraetions were subjected to thin layer chromatography, the major classes again were triglycerides and cholesterol with very faint bands corresponding to free fatty acids, cholesteryl esters and diglycerides. The results for the analyses of the fatty acid methyl esters from the triglyceride fractions are shown in Table 2, There were no significant difterences between the normal and dystrophic samples. The other neutral lipid elasses were similarly treated but. because of their very low concentrations, the results were unreliable; however, there were certainly no obvious difTerences between the normal and dystrophic samples. TABLE 2 Fally acid composition aj triglyceride. Fatty acid

14.0

16:0

16:1

18:0

18:1

1S:2

Normal Dystrophic

2-5 + 0-1 2-l±0-l

23-8+ 1-7 22-0 + 0-8

9.6 + 0-9 7-2±l-5

4-3±0-2 4 5+0 4

46-5±l-2 50-5 + 2-2

13-2 + 0 6 13.6±l-0

Each value is expressed as a percentage ol the total tattv acid eomposition. Values are given as means + S.E.M. for4 analyses.

Pho.sphoUpids

Analyses ofthe phospholipid compositions of normal and dystrophic muscle showed a significant decrease in the choline phosphoglyceride fraction which could contain phosphatidylcholine and the plasmalogen. phosphatidylcholine (Table 3). However, the concentration ofthe plasmalogen must be very low, since only trace amounts of dimethylacetals were found in this fraction (Table 4). Hence, the observed decrease must be in phosphatidylcholine. The amount of lysophosphatidylcholine is low in both samples and is probably a degradation product. Significant changes were not seen in the proportions of the other phospholipids. TABLE 3 Phuspholipidcompi'sititin ol normal anddyslrophic mouse muscle.

Choline phosphoglyceride Ethanolamine phosphoglyceride Sphingomyelin Phosphatidylmositol + Phosphalidvlserine Cardiolipin Lysophosphalidylcholine

Normal

Dystrophic

P

56 2±O-8 26-2+ l-l 3-0 + 0-6

50-9±0-6 30-0+1-8 4 l±l 0

b - i : 30 ad 00 00 6 r-1 n

MUSCLE LIPIDS IN DYSTROPHIC MICE

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0.

C

c r

C

• , «

c

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s

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0

0

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+ 1 -fl

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OH

7.

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f

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-n

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Normal

Lysophosphalidylcholine

n.s. n.s.

Dystrophic

a.

n.s. n.s. . University of Western Australia, and Department of PalholoL>, King Edward Memorial Hospital for Women, for the use ofthe gas chromalograph and for helpii.: advice. The financial assistance of the Muscular Dystrophy Research Association of Western '\Lislralia(Inc.) is gratefully acknowledged.

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'Abnormalities o! pciipheral .;cr\es in murine muscular Jv>-iii>phy,",/ .witrol Sci.. 18,227-247.

BLRR, L, H.. and MCLENNAN. H. (1961): 'The

transport of potassium ions in normal and dyslrophic mouse muscle.',/, Phy\iai.(Liiud.). 158.324-332.

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P. H. PEARCE AND B. A. KAKULAS

DEI KRF.TstR. T. A., and L i v t r i . B, G. (1977): "Skeletal muscle sarcolemma from normal and d\strophic mice," Hiochcni. / , 168, 229-237, FOUH. J.. LKKS. M . . and SLOANF STANLEY. G ,

H. (1957): 'A simple method for the isolation and purification of total lipids from animal tissues,',/. «/rj/ f7;rH!., 226.497-509, HuuHts. B, P. (1965): 'Pliospholipids in normal and dystrophit mouse muscle," In "Research in Muscular Dystrophy. Proc, III S\mposium". Pitman Medical Publishing Co. Ltd.. London. pp,187-194, KuKSis. A.. MARAI. L,. and GuRN\Lt,. D, A, (1967): "Direct gas chromatographic examination of total lipid extracts-",/. Liptd Rev, 8. 352-358,

MORRISON. W , R , . and SMITH. L, M , (1964):

'Preparalion of fatty acid methyl esters and dimethylacclals from lipids with boron nuoride-methanot."./. LipidRe\..5, 600-60S. OWENS. K . (1966): 'A twn-dimensional thin layer chromatographic procedure for the estimation of plasmalogens.' Biochem. J.. 100. 354-361. OWENS. K , (1968): 'Fatty acid and aldehyde composition of normal and dystrophic muscle lipids.' In ""Research in Muscular Dystrophy, Proc. IV Symposium". Pitman Medical Publishing Co. Ltd., London, pp, 363-37!. OWENS. K , , a n d HICIHES, B, P. (1970): 'Lipidsof

KUKSIS. A,. STACHNYK, O , . and HOLUB. B . J,

dystrophic and normal mouse muscle: whole tissue and particulalc Tractions."/ tJpidRes., 11.486-495,

(1969): "Improved quanlilation of plasma lipids by direct gas-liquid chromatography," J. Lipid Res.,\Vi,bH)-bbl.

PiisiiKhR. G. A., and Api'LL. S, H,(I98O): "Red blood ceil alterations in muscular dystrophy: the role of lipids.' .Mu.scte und Nerve. 3. 70-8 1,

KWOK. C , T , . and ALSTIN. L. (1978): "Phospho-

lipid composition and metabolism in mouse muscular dystrophy,' Biochem. ./,. 176. 15*22, KWOK, C , T , . KL FKER. A. D,. TANG. B, Y . . and

AUSTIN. L. (1976): "Phospholipid metabolism in murine muscular dystrophy,' /:,v/'. Senrol. 50,362-375, LILIENTHAL. J, L,. JR,. ZIERLEIH. K , L . . FOLK. B,

P.. BuKA. R., and RiLti. M. J. (1950): "A reference base and system for analysis of muscle constituents,' J. Biol. Chem.. 182. 501-508, LOWRY. O, H,. Rosf-iiRotciH. N, J,. FARR. A, L,.

and RANDALL R , J , (1951): "Protein measurement with the Folin phenol reagent,',/. Biol. Chem..l9i.l65-215. MACPIKE.

A , D , . and

MEIFR.

H , (1976):

"Comparison of dy and dy'K two alleles expressing forms of muscular dystrophy in the mouse (39283).' Proc. Soc. E.xp. Biot. .\ted. 151.670-672, MARTONOSL A , (1968): "Sarcoplasmic reticulum. Vi, Microsomal Ca* ' transport in genetic muscular d>strophy of mice." Proc. Soc. E.xp. Biol. Med., 127, 8i4-828.

S H U L L R , L . . and ALFIN-SLATHR. R . B . (1958):

"Tissue lipids of Dysirophia muscularis. a mouse with inherited muscular dyslrophy." Prac. St)c- E.xp. Bit)!. ,l/f(/. 97.403-405, SNi;nt-: