Ultrastructure of dystrophic mouse sarcoplasmic reticulum

BIOCHEMICAL

19, 277-293 (1978)

MEDICINE

Ultrastructure of Dystrophic Sarcoplasmic Reticulum ROBERT E. MRAK'AND Department

of Zoology.

University

of

Received

July

R.J.

Calijbrnicl, 27.

Mouse

BASKIN Davis.

Californicl

95616

1977

Ultrastructural alterations in the membranous systems of genetically dystrophic animals and man have been consistently described (l-5). In the dystrophic mouse, swelling of the sarcoplasmic reticulum (SR) is evident at a very early age (6-S). Recent freeze-fracture studies on isolated dystrophic chicken SR have shown a decrease in intramembranous particle density (9, IO), a change which has also been observed in erythrocytes of dystrophic chickens (1 I). The significance of these changes. however, remains controversial, particularly in light of a recent study showing swelling of the T-tubule system, which has a relative paucity of such particles normally, in the dystrophic chicken (12). In a previous paper (13) we described functional alterations in the calcium transport activity of the sarcoplasmic reticulum from genetically dystrophic mice. These alteration may form a link in a pathogenetic sequence where an alteration in the membranous lipid structure results in the observed functional deficits which in turn result in the mechanical alterations seen in these muscles. In this study we examine the ultrastructure of sarcoplasmic membranes, both in the intact muscle and in isolated preparations, for the purposes of correlating pathological structure with pathological function and of comparing findings in the mouse with those in the chicken. MATERIALS

AND METHODS

Homozygous 129B6Fl dydy mice were used as described in a previous paper (13). The preparation of isolated sarcoplasmic reticulum vesicles was also carried out as described previously (13). ’ Present Nashville.

address: Tenn.

Department

of Pathology.

School

of Medicine.

Vanderbilt

University.

37232. 277 0006-2944/78/0192-0277$02.00/O Copyright @ 1978 by Academic Press. Inc. All right? of rsproductmn in any form reserved.

MRAK AND BASKIN

278

Mice were sacrificed by cervical dislocation and the hind limb muscles rapidly exposed and fixed in situ with 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. for I hr at 0°C. The muscles were then washed twice in ice-cold 0. I M phosphate buffer. and small pieces of tissue were cut from superficial layers. These were postfixed for I hr in 2% 0~0, at PC, dehydrated through a series of graduated acetone: water mixtures. and embedded in an Epon-Araldite mixture. Sections were stained for IS min. using 2% hot alcoholic uranyl acetate stain (14) and poststained with lead citrate (IS). SR vesicles were centrifuged to a pellet and then fixed and further processed as described above. Free:<> jhctllrirzg. Mice were sacrificed, and the hind limb musculature was exposed and fixed with glutaraldehyde as described above. The muscles were then soaked in 50% (by volume) glycerol in phosphate buffer overnight at 0°C. Small pieces were then cut from superficial muscle layers, placed on 3-mm cardboard or metal disks. rapidly frozen in liquid Freon. and placed in liquid nitrogen. Freeze fracturing and carbon-platinum shadowing were performed following the technique of Deamer and Baskin (16) in a Balzers freeze-etching device at a temperature of - 100°C and a pressure of 4 x IO + Tort-. Isolated unfixed vesicles were suspended in 30% (by volume) glycerol in mouse SR buffer. centrifuged to a pellet, and carved into small hills on a 3-mm disk for freezing and fracturing. Negcrtil)c stcrining. A drop of the appropriate microsomal suspension was placed on a Formvar-coated electron microscope grid for a few seconds and then siphoned off by touching the grid with a piece of dry filter paper. A drop of the appropriate negative stain (2% aqueous uranyl acetate or 2% aqueous phosphotungstic acid) was then applied in the same manner. Electron microscopy. All specimens were examined in a Hitachi HU-I IE electron microscope at an accelerating voltage of 7SkV. Strrtistics. Differences between corresponding values were assessed for significance using a two-tailed Student’s t test. Thin srctioning.

RESULTS Ultmstrmc.ture

of Normnl

Mouse Mrrscle

Freeze-fracture replicas of normal mouse muscle show regularly spaced and well-aligned sarcomeres (Figs. I and 2). The A, 1. and H bands are readily apparent. The Z line is quite straight and regular. The SR is interrupted by two T tubules per sarcomere, one at each A-I junction. The freeze-fracturing process cleaves biological membranes along their inner hydrophobic plane (17) as the membrane’s hydrophobic forces are much weaker than the polar interactions of frozen water. The exposed faces are thus the inner lipophilic aspects of one or the other of the

DYSTROPHIC

FIG.

Normal

1.

are evident fractures FIG. 2. leaflet (SR (T)

are

mouse

and the H zone is from below.

also

Normal PF) and particulate.

SARCOPLASMIC

muscle. can

The

be seen.

mouse muscle freeze particle-poor luminal M =

mitochondrion.

Z lines Bar

179

RETICULUM

are

straight

= I .O pm.

and

Direction

regular.

The

of shadowing

fracture. Portions of the particle-rich leaflet (SR EF) of the SR are visible. Bar

= 0.5

A and

I band\

on all freeLr cytoplasmic The T tubules

pm.

membrane’s two lipid monolayers. These are designated by convention “PF” faces (the cytoplasmic leaflet) and “EF” faces (the luminal leaflet). The cytoplasmic leaflet of the SR is studded with 8 to 9-nm particles (Fig. 3) which represent the calcium transport protein of SR as originally postulated by Deamer and Baskin (16). The particles are present at a density of approximately 3000 particles/ pm “. The luminal face of the SR is quite smooth. Arrays of parallel ridges or rod-like structures, also 8-9 nm in width, are often seen running at right angles to the axis of the sarcotubular lumen. These ridges have a crest-to-crest spacing of about 20 nm in the cisternal portion and may be as close as IO nm in the longitudinal portion of the reticulm. The T tubules are also particulate. with particles of I I to 13-nm diameter. These are seen predominantly on the luminal leaflets, with a density of about 1800 particles/ pm “. The cytoplasmic leaflet displays occasional pits (Fig. 3).

280

FIG. 3. (EF) leaflets

MRAK

Detail of normal are shown. Pits

Bar = 0.2 pm. (b) Cisternal shuwn. PF = SR cytoplasmic

mouse (circles) ridging leaflet.

AND

BASKIN

muscle triad. (a) The SR cytoplasmic are visible in the cytoplasmic leaflet (arrows) Bar =

and I I- to 0.2 km,

13.nm

T tubule

(PF) and luminal of the T tubule (T). (T)

particles

are

Dystrophic fibers were found in all stages of involvement from apparent normality to near total loss of fine structure. In Fig. 4 and 5 the SR is seen in varying stages of dilatation and vesiculation. The myofibrillar apparatus is uninvolved except for occasional irregularities of the Z line and mild nonalignment of ad.jacent sarcomeres. T tubules are often recognizable in

DYSTROPHIC

FIG. 4. Dystrophic swollen and vesiculated

SARCOPLASMIC

mouse muscle. Normal SR. A normal-appearing

RETICULUM

appearing T tubules (arrows) mitochondrion is seen adjacent.

381

persist amid Bar = I .0 Km.

282

MRAK

AND

BASKIN

the midst of extensively dilated and fragmented SR and always appear quite normal. In Fig. 5 a T tubule is seen apparently uninvolved in an area where the SR shows extensive swelling. Intact mitochondria are also evident in these sections. Freeze fractures of dystrophic mouse muscle further defined the ultrastructural abnormalities. In areas where the SR was not swollen or fragmented, no abnormalities were noted by this method. Such “uninvolved” SR displayed the 8 to 9-nm particles noted in normal muscle on the cytoplasmic leaflet and at a normal density (Fig. 6). The T tubules possessed I I to l3-nm particles on their luminal leaflets, again at normal density. The ridges on the SR cytoplasmic leaflets noted in the normal mouse were present both in the cisternal and longitudinal portions of the reticulum (Fig. 6). Areas of SR swelling, however, consistently showed loss of particles (Fig. 7). Swollen or vesiculated membranes possessing particles in normal density were never observed.

Thin sections of the four recovered fractions demonstrated that all were predominantly vesicular. Vesicle diameters ranged from 20 to 800 nm. Vesicles from F20 exhibited the greatest heterogeneity of sizes, while the middle two fractions were relatively uniform (Fig. 8). Although tubulelike forms were occasionally noted, no structures resembling triads were seen. In the fourth fraction, various contaminants were noted: cristaecontaining mitochondrial remnants. apparent nuclear fragments, and fibrillar debris. None of these structures was noted in any of the other fractions. Negative staining with phosphotungstic acid demonstrates collapsed vesicles, as described by Ikemoto et al. (18) in all fractions (Fig. 9). The tadpole shape of the vesicles has been shown to be an artifact (16) due to the hyperosmolarity of the stain (19) and is not seen in preparations negatively stained with uranyl acetate (Fig. IO) as described by Deamer and Baskin ( 16). High magnification of negatively stained vesicles demonFIG. flanked

5.

Dystrophic

by cisternal

SR.

mouse

muscle.

which

communicates

A normal-appearing (small

T tubule arrow)

with

(large

a dilated

arrow)

vesicle.

is seen Bar

= 0.5

pm.

FIG.

6.

vesiculation leaflets (arrows).

are

Dystrophic and swelling.

mouse muscle Normal-appearing

freeze

fracture. This SR cytoplasmic

visible. Ridging is present in the longitudinal The T tubules (T) display I I- to 13-nm particles

area is free (SR PF) and

and cisternal on their luminal

of membranous luminal (SR EF)

portions leaflets.

of the SR Bar = 0.5

v. FIG. 7. Dystrophic mouse muscle freeze fracture. Swollen SR elements cent to normal appearing T tubules (T). The particle density of the cytoplasmic swollen SR varies from near normal (single arrow) to nearly smooth (double mitochondrion. Bar = 0.5 brn.

are seen adjaleaflets of the arrow). M =

DYSTROPHK

FIG. normal FIG.

8. Thin F35, (D) 9.

sections dystrophic

SARCOPLASMIC

RETICULUM

of isolated SR fractions. F3.5. Bar = 0.5 pm.

Phosphotungstic

acid

negative

stain

(A)

Normal

of isolated

FX.

183

(6)

SR showing

dystrophic osmotic

FIX. collapse

3- to 4-nm surface particles. (A) and (C), normal: t B) and CD). dystrophic. 0.S pm: tC’) and tD). bar = 0.1 Fm, FIG. IO. Uranyl acetate negative stain of isolated SR showing round

t A) and

particles.

(C).

(A).

normal:

tB) and

(0.

dystrophic.

(A)

and

(B).

bar

= 0.5 ym;

shape

(B). and

bar

(C) and

bar surface

= 0. I pm.

=

284

MRAK

AND

BASKIN

strates the 3 to 4-nm surface particles associated with the SR adenosine 5’-triphosphatase (ATPase) (16, 18, 20, 22). Freeze fractures of isolated microsomal fractions are shown in Fig. 11 and 12. The dominant vesicle in F20 was large and devoid of particles. These vesicles reached diameters of 800 nm. In the remaining fractions vesicle sizes were much more uniform, generally in the 50- to 200-nmdiameter range. Four fracture faces accounted for 95% of the exposed membrane area: (i) a smooth convex fracture face: (ii) a convex fracture face containing I I- to 13-nm particles at a density of 1900 particles/ prnZ; (iii) a smooth concave fracture face: and (iv) a concave fracture face containing 8-nm particles generally at a density of 2500-3100 particles/ pm’. The particulate faces are illustrated in Fig. 13. Tables I and 2 give the distribution of these fracture faces for the three heavier fractions. both as a percentage of vesicles present and as a percentage of total membrane area. In F26. one-third of the convex fracture face area is particulate, while two-thirds are smooth. For the concave faces. two-thirds of the exposed area are particulate, and onethird is smooth. This reciprocal relationship suggests that the membranes in this fraction are particulate on one leaflet only. with the smooth convex faces corresponding to the particulate concave faces and vice versa. This is supported by the vesicle counts showing 7S% of the convex vesicles to be smooth and 74% of the concave vesicles to be particulate. Conversely, 32% of the convex vesicles are particulate and 247~ of the concave vesicles are smooth. A similar correlation was found in regard to mean vesicle diameters: for convex fractures, the smooth vesicles are smaller, and for concave fractures. the smooth vesicles are larger than the particulate ones. The third fraction gives very nearly the same profile as the second; there are slight increases in the convex particulate and concave smooth portions. both in vesicle number and total area. and corresponding decreases in the convex smooth and concave particulate portions, but these changes are not significant. Mean vesicle diameters show the same pattern as F26. The fourth fraction shows a considerable increase in smooth fracture faces. both concave and convex, over the middle two fractions. This is consistent with an additional population of vesicles containing no particles at all. Indeed the existence of such vesicles is mandatory since smooth faces comprise over half of both the exposed concave and convex areas in this fraction. If the convex particulate and the concave particulate faces are accepted as representing two distinct vesicle populations, then yield ratios for these two populations can be calculated. Taking into account the relative yields of the microsomal fractions (13). an overall ratio of 2.3: I can be calculated for concave particulate area: convex particulate arca.

DYSTROPHIC

FIG. I I. Isolated F20 microsome 0.5 fim. FIG. 12. Isolated F16 microsome late and smooth faces are seen. Bar FIG. 13. Detail showing concave microsomes. Bar = 0.1 Wm.

SARCOPLASMIC

freeze

fractures.

185

RETICULUM

(A) Normal.

(B) dystrophic.

Bars =

freeze fracture. (A) Normal. (B) dystrophic. Particu= 0.5 pm. and convex particulate fracture faces of isolated F16

286

MRAK

AND BASKIN TABLE

PARTICLE

DISTRIBUTION

I

OF CONVEX

FRACTURE

Smooth

FACES”

I I- to 13-nm Particles

Total vesicles counted

Vesicles (5%)

Area 6%)

Vesicles (c/c)

Area (Q)

Normal F26 Dystrophic F26

355 736

75 t 6 63 c 2

68 f 9 65 + 4

22 t 6 37 t 2

28 2 8 33 + 5

Normal F35 Dystrophic F35

146 406

61 2 5 76% I’

64 2 4 75 2 3

34 t 4 22 +- I*

31 t5 22 + 3

Normal F44 Dystrophic F44

139 126

88 t 2

86 t 2

77 2 8

87 + 5

ltl 24 ? 8

6 t 0.4 13 -+ 5

” Values are means of three (for F26 and F35) or two (for F45) separate determinations + SEM. * Difference from corresponding normal values is statistically significant (P ~0.05).

Ultrastructure

of Isolated Dystrophic SR

Thin sections of pelleted fractions from dystrophic mice showed that all fractions were predominantly vesicular (Fig. 8). As with normal fractions, F20 was found to contain an abundance of very large vesicles, while fractions F26 and F35 appeared more homogenous. The heaviest fraction consisted of quite a heterogenous accumulation of cellular debris and appeared somewhat less vesicular than the corresponding normal fraction. Mitochondrial remnants were noted but were far from dominant. as was true of the normal fraction. TABLE PARTICLE

DISTRIBUTION

2

OF CONCAVE

FRACTURE

Smooth

FACES”

8- to 9-nm Particles

Total vesicles counted

Vesicles m

Area (5%)

Vesicle 1%)

Area (%)

Normal F26 Dystrophic F26

359 569

24 + 5 20 k 5

29 t 8 34 -c 6

74 2 4 80 k 5

66 + 7 66 k 6

Normal F35 Dystrophic F35

414 480

37 t 2 32 k 2

39 k 3 482 I*

63 + 2 68 2 2

61 t 3 51 f 2’

Normal F44 Dystrophic F44

220 224

58 48

64 k 4 66 f 4

42 c II 53 t IO

36 + 4 34 t 4

k IO k IO

I’ Values are means for three (for F26 and F35) or two (for F44) separate determinations SEM. * Difference from corresponding normal values is statistically significant (P ~0.05).

+

DYSTROPHIC

SARCOPLASMIC

287

RETICULUM

Negative staining of dystrophic fractions did not reveal any significant differences from normal microsomes (Figs. 9 and IO). Particulate fringes as described for normal SR were noted in all fractions. Phosphotungstic acid stains caused osmotic collapse of vesicles while uranyl acetate did not, in accordance with observations on normal vesicles. Freeze fractures of dystrophic vesicles are shown in Figs. 9 and IO. The dominant fracture faces were the same ones noted for normal vesicles: convex faces with 1 I- to 13-nm particles at density of 1900 particles/ pm “, concave faces with 8- to 9-nm particles at a density of 3 100 particles/ pm “. and smooth faces. The relative proportions of these faces are given in Table I. In all fractions except F35 the relative abundance of the various fracture faces is identical to the findings in normal vesicles. in F35 an increase in concave smooth fracture face area and a corresponding decrease in concave particulate fracture face area are evident. An increase in convex smooth face area is also present, but this does not achieve statistical significance. Since significant differences in yields of fractions F3S and F44 exist between normal and dystrophic preparations (131, corrections for such yields should be made in comparing these fractions with their normal counterparts. This calculation is performed in Table 3. Thus, in terms of absolute yield, there is a highly significant increase in TABLE CALCULATED

3

TOTAL YIELDS OF VESICLES RERPESENTED BY VARIOUS FREEZE-FRACTURE FACES” Convex faces Smooth

Concave faces

II- to 13-nm Particles

Smooth

8- to 9-nm Particles

Normal F35 Dystrophic F35

273 ” 16 467 YZ l8***

130 k 22 136 2 I9

166 + I2 298 2 8”*”

258 2 I I 319 -c IO”*

Normal F44 Dystrophic F44

195 2 5 70 a 4***

I3 % 1 IO 2 4

I45 + 9 51 t 3**

82 -c 9 26 k 3-

Normal: Total yield Dystrophic: Total yield Norma1 total/ dystrophic total

689

227

433

523

737

223

481

497

1.07

0.98

I.11

0.95

‘I Values, in micrograms per gram of muscle, are expressed as means 2 SEM of three (F35) or two (F44) determinations. * P < 0.05. significantly different from corresponding normal value. ** P < 0.02, significantly different from corresponding normal value. *** P < 0.01, significantly different from corresponding normal value.

288

MRAK AND BASKIN

both smooth convex faces and smooth concave faces in dystrophic F35. A lesser increase in concave particulate faces is seen. while convex particulate yield is unchanged. Complementary changes are seen in fraction F44 such that total yields for the various fracture faces are very similar in normal and dystrophic preparations. These data are compatible with the conclusion that the 200 pg of increased yield (13) in dystrophic F35 corresponds to material found in fraction F44 in normal preparations. Moreover, two-thirds of this “transfer” consist of vesicles that are particle-free and one-third consists of vesicles that contain 8- to 9-nm particles associated with their concave leaflets and no particles associated with their luminal leaflets. Vesicles containing 1 I- to 13-nm particles on their luminal leaflets are not changed in their distribution among the dystrophic fractions. Overall a slight increase is seen in smooth fracture faces (7- 11%). When the yields of convex particulate vesicles and concave particulate vesicles are compared, the overall ratio is 2.2: I, the same as for normal preparations. This is in accord with the data in Table 3, which suggests that differences between normal and dystrophic fractions are due primarily to “shifts” of material between fractions.

DISCUSSION The freeze-fracture micrographs presented here represent the first such study of mouse muscle. The sarcoplasmic reticulum was found to exhibit 8- to 9-nm particles at high density and primarily on the cytoplasmic leaflet. Such particles have been described in SR from various sources (12, 23-28). with the same asymmetry of distribution and high density of particles. The presence of these particles in vesicles reconstituted with purified preparations of the SR (Ca’+ + Mg’+)-ATPase (29,30) has confirmed their indentification as the calcium pump protein (16). Glutaraldehyde, used as a fixative in this study, is known to produce no artifacts of intramembranous particle structure or distribution (3 I-36). The T tubules of mouse muscle are also particulate. Particles in the size range noted here (I I- 13 nm) have been described in the T tubules of frog and fish fibers, although at a density less than that observed here (26). Particles of about IO-nm diameter have been described in the fractured T-tubule faces of the tarantula spider (25) and rat (28). While these particles were found in low density and predominantly on the cytoplasmic leaflet in the tarantula, they were described as “numerous” and predominantly luminal in the rat.

DYSTROPHIC

Ultrrrstructure

of Dystrophic

SARCOPLASMIC

RETICULUM

289

Mouse Muscle

Thin sections of dystrophic mouse muscle obtained in this study are in agreement with earlier such studies in both man and mouse. Dilatation and fragmentation of SR was an early finding, being preceded only by Z-line abnormalities, as described by Hudgson and Mastaglia (37). SR swelling and fragmentation was evident only early in myofibrillar degeneration. With advanced involvement, only small vesicular remnants of the SR are seen, and in many fibers little or no change is apparent. It is therefore quite difficult, in the absence of careful morphometric analysis. to make any definitive statements regarding the overall membranous content of normal vs dystrophic muscles. The T tubules, however, were consistently spared until the disease reached a late stage in contrast to recent findings in dystrophic chickens (12). Freeze fractures of dystrophic muscle demonstrate a paucity of particles in areas of SR swelling. Since the particle density and distribution in areas free of swelling were normal, it appears that SR swelling involves either an addition of particle-free membrane or a concomitant loss of particles. Another possibility is that ATPase molecule, which may exist in the membrane as an oligomer (38), dissociates with SR swelling and is, as a consequence, no longer demonstrable ultrastructurally. Malouf and Sommer (12) have recently shown that at least some of the swollen membranous vesicles observed in muscular dystrophy of the chicken (I) are derived from T tubules on the basis of a freeze-fracture study. This is confirmed by observations made in our laboratory (L. Crowe and R. J. Baskin, unpublished). The present results suggest that this is not the case in muscular dystrophy of the mouse; evidence of SR swelling is unmistakeable (Fig. 5), while normal-appearing T tubules were identifiable in all but the most severely affected fibers, in which only small vesicular remnants of uncertain origin were present. This may reflect a basic difference between the murine and avian diseases. Origin und Purity of Isolated Fructions. Fractions F26 through F44 all appear to consist in large part of vesicles derived from the sarcoplasmic reticulum. Thin sections of all three fractions show a predominately vesicular makeup. Negative stains reveal the 3- to 4-nm “surface particles” which are found in isolated SR from mouse ( 18). rat, frog (20), and rabbit (16). as well as purified preparations of SR ATPase (21, 22). Freeze fractures of the isolated fractions show that 36-66s of the membrane area contains the 8- to 9-nm particles found in the SR in situ (Table 2). On the basis of the freeze-fracture data presented in Tables I and 2. we can conclude that two-thirds of the membrane area in fractions F26 and

290

MRAK

AND

BASKIN

F3.5 are derived from the sarcopfasmic reticulum, and one-third has a different origin. The particle size. density, and asymmetry of the nonreticular vesicles are quite similar to those of the T tubules in situ. Thus it may be that at feast part of these vesicles are derived from the transverse tubular system. Luff and Atwood (39) found a volume ratio of 13: I for the sarcopfasmic reticulum and T-tubule system in mouse extensor digitorum longus and sofeus muscles. A ratio for membrane areas is not given. but if we assume similar surface-to-volume ratios in the two systems, a membrane area ratio of 5: I can be calculated. These figures are very close to the values found by Peachy (40) for frog sartorius. In the present study a ratio of 2.3:1 was found for the membrane areas of recovered vesicles bearing S- to 9-nm particles on their external leaflets and recovered vesicles bearing I I- to f3-nm particles on their fuminaf leaflets, which is about half the expected ratio if the fatter vesicles are derived from the T-tubule system. It may be that T-tubule fragments are easier to extract from muscle than SR fragments. Meissner (41) isolated light and heavy fractions of rabbit SR, corresponding roughly to our F26 and F33, on a sucrose density gradient. He found the heavy vesicles to be filled with electron-dense material similar to that found in the terminal cisternae of intact rabbit muscle and concluded the heavy vesicles originated from the terminal cisternae and the fight ones from the longitudinal elements of the SR. This ultrastructural difference between heavy and fight fractions is not apparent in the present study. F44 contains a substantial proportion of heavy SR vesicles but contains in addition miscellaneous cellular debris. The four microsomaf fractions isolated from dystrophic mouse SR appear to correspond very closely to those found in normal preparations. Dystrophic F26 does not differ noticeably from its normal counterpart in ultrastructural appearance (Figs. 8, 10, and 17: Tables I and 2) or total yield (I3). In fraction F35 the total yield is somewhat higher (13). and there is a relative increase in smooth concave fracture face area and a relative decrease in particulate concave fracture face area (Table 2). These differences can be accounted for by the assumption of a “transfer” of 200 pg of microsomaf yield/g of muscle from F44 to F35. two-thirds of which consist of vesicles that are particle-free and one-third of which consists of vesicles that are concavely particulate (Table 3). Thus F3S in dystrophic preparations appears to consist of material referable to the same fraction in normal preparations as well as a sizable amount of material referable to F44 of normal preparations, due to the lesser buoyant density of this material. Fraction F44 in the dystrophic, as in the normal, appears to contain a great degree of miscellaneous cellular debris. This debris appears to be of greater relative proportion due to the “transfer” of much material to F35.

DYSTROPHIC

SARCOPLASMIC

RETICULUM

391

It is of interest that the overall yield of microsomes is not significantly increased (13) while the yield of smooth fracture faces is increased only slightly if at all (Table 3). This is in marked contrast to findings in the dystrophic chicken where total microsomal yield is more than twice that in normal birds (1) and a significant increase in smooth fracture faces is seen in isolated fractions (9, 10). some of which may represent T tubules (12). The dystrophic chicken, however, manifests marked swelling of membranous organelles (l), far beyond that seen in the dystrophic mouse. Furthermore. although SR swelling is a consistent finding in dystrophic mouse muscle, it is not ubiquitous, and fibers with advanced involvement show only small vesicular remnants while many fibers may appear normal or near normal. It is further observed that the concave particulate vesicles were unchanged in their yield and position in the gradient (Table 3). These vesicles are apparently unaffected by the dystrophic process. This is in accord with the suggestion that these vesicles may represent T-tubule fragments and with the apparent lack of involvement of the T tubule in the dystrophic process. There were no other ultrastructural differences between normal and dystrophic SR. In particular, isolated vesicles displayed normal response to the osmotic effects of negative stains (Figs. 9 and IO) in contrast to the findings of Sreter et al. (42). who described osmotic collapse of normal, but not dystrophic, SR vesicles when negatively stained with phosphotungstic acid. The greater permeability of dystrophic vesicles thus found in their study could represent vesicle damage incurred during isolation or a greater representation by vesicles from muscles with advanced involvement in the dystrophic process (the ages of mice used in their study is not mentioned.). SUMMARY A freeze-fracture study of normal mouse muscle revealed the following ultrastructural features of the sarcoplasmic reticulum (SR) and the T-tubule system: The SR cytoplasmic leaflets possess 8- to 9-nm particles at a density of 3000/pm2. The SR luminal leaflets possess few particles. The T tubules possess II- to 13-nm particles, primarily on the luminal leaflet, at a density of 1800/pmZ. Thin sections of dystrophic mouse muscle showed areas of SR dilatation and fragmentation. The T tubules were uninvolved until late in the disease process. Freeze fractures showed these dilated areas to be relatively particle-free. Areas of SR free of swelling or fragmentation did not differ from normal in freeze-fracture ultrastructure. Dystrophic mouse muscle T tubules displayed no abnormalities in freeze-fracture replicas. Enriched preparations of fragmented SR vesicles were isolated. On a sucrose density gradient, light SR from dystrophic mice was shown to be

292

MRAK

AND BASKIN

of purity equal to the normal preparation by ultrastructural (thin section, freeze fracture, and negative stain) criteria. Differences in yield of the heavier fraction were shown to be explainable by postulating a subpopulation of vesicles with increased buoyancy. The purest fraction appeared to consist of sarcoplasmic reticulum vesicles (about two-thirds of the material) and vesicles from another source. possibly T tubules. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 3 I. 32. 33. 34.

35. 36.

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