J Cell Set. 6, 593-603 (1970) 593

Printed in Great Britain

THE PROLIFERATION OF MYOFIBRILS

DURING MUSCLE FIBRE GROWTH

G. GOLDSPINK

Department of Zoology, Unwerstty of Hull, England

SUMMARY

Myofibrils in muscle fibres of different sizes and different ages were examined and measuredusing phase-contrast and electron microscopy. During the post-natal growth of the mousebiceps brachii muscle the number of myofibnls in some fibres increases from about 75 to 1200The range of myofibnl size was from 0-4-1 2 fim. The distribution of myofibnl sizes in musclesof all ages studied was bimodal

A high incidence of longitudinal splitting of myofibrils was observed with the electron micro-scope in differentiating muscle fibres and in some medium and large muscle fibres. Sizemeasurements with the electron microscope showed that the splitting myofibrils were abouttwice as large as non-splitting myofibrils and that the myofibrils split more or less down themiddle. A possible explanation for the splitting is that the peripheral I filaments are pulled at anangle slightly oblique to the myofibnl axis, because of the discrepancy in the A and I-filamentlattice spacings. When the myofibnl reaches a certain size the oblique pull of the peripheral Ifilaments is strong enough to cause the Z disks to rip.

From data on the size, shape and number of myofibnls at different stages of growth it wasconcluded that longitudinal splitting is the means by which the number of myofibrils increasesduring post-natal growth.

INTRODUCTION

The post-natal growth of most striated muscle fibres of the laboratory mouse takesplace in two stages. The first of these stages can be regarded as the completion ofdifferentiation. The mouse is born in a relatively immature condition and differentia-tion of the musculature continues for 2 or 3 weeks after birth (Rowe & Goldspink,1968). During this time the myotubes, which are about 100 fim2 in cross-sectionalarea, develop into small muscle fibres which contain many more myofibrils and measureabout 325-500 fim2 in cross-sectional area. The growth of the fibre stops for sometime when it reaches this stage; this may be regarded as the basic level of muscle fibredevelopment (Rowe & Goldspink, 1968), or the 'small phase' (Goldspink, 1962, 1964,1965). In some muscles of the mouse, for example, the soleus and extensor digitorumlongus, the fibres stay at this level of development throughout the animal's life.However, in other muscles some of the small fibres undergo further growth (hyper-trophy) until they attain a size of approximately 1250 /tm2. This further growth isthe second stage of post-natal muscle fibre growth and is a particularly interestingprocess because it is the means by which a muscle can adapt its size and strength tothe function it has to perform (Goldspink, 1964).

The number of 'small phase' fibres that undergo further growth depends on the

594 G. Goldspink

work load the muscle is subjected to and the age and nutritional status of the animal(Goldspink, 1962, 1964; Rowe & Goldspink, 1968, 1969).

The minimum time for a ' small phase' fibre to develop into a ' large phase fibre' hasbeen shown to be about 1 \ days. This is a surprisingly short period, as it has also beenshown that the increase in size of the fibre results from a large increase in its myofibrilcontent. The increase in the myofibril content of the fibre is due to an increase in boththe number and the size of the myofibrils (Goldspink, 1965). This previous study posedthe interesting question of what mechanism is involved in the proliferation of myo-fibrils during differentiation and further growth (hypertrophy) of the muscle fibres,and how the myofibrillar content of a fibre can increase so much in so short a time. Thework described here was an attempt to answer these questions by examining musclefibres at different stages of growth with the phase-contrast and electron microscopes.

MATERIALS AND METHODS

The mice used were all males of the I2g/Re strain which was originally obtained from theJackson Memorial Laboratories, Bar Harbor, U.S A. They were maintained on a modified dietformula 41 b (Oxoid Ltd) with food and water available at all times. The biceps brachu musclewas the muscle chosen for this study as it has previously been shown that approximately 40 % ofits fibres have undergone further growth by the time the mouse is half grown (Goldspink, 1962;Goldspink & Rowe, 1968; Rowe & Goldspink, 1969). Other considerations were that thismuscle has a relatively simple, fusiform structure, with fibres running the full length of themuscle belly, and it is small enough to be fixed whole and in situ.

Light microscopy. The muscles used for the light-microscope measurements were prepared inthe following way. Mice of different ages were killed by dislocation of the cervical vertebrae,the forelimbs were skinned and severed from the carcass and then pinned in the fully extendedposition to a strip of cork The forelimbs were then immersed in Flemming's-without-aceticfixative (F.W.A.) for 5 h. The muscles were dissected from the limbs and replaced in the fixativefor a further 25 h. Following this they were washed for 12 h in running water and dehydratedin ethanol and embedded in ester wax (Steedman, i960). Serial transverse sections were cutat 4 fim, stained in Heidenhain's iron haematoxyhn and mounted in Canada Balsam. Sectionstaken from the middle of the muscle belly were examined with a Leitz Ortholux microscopeusing phase-contrast optics. Fibres of different sizes in which the myofibrils could clearly be seenwere photographed, and the negatives enlarged to give a final magnification of x 3750. The totalarea of each fibre was measured using a planimeter. The diameter of the 10 myofibrils of eachfibre was measured using micrometer calipers under a large magnifying lens ( x 25). The totalnumber of myofibrils within the fibre was obtained by counting each individual myofibril,using an electrical counting pen.

Ten fibres were selected and measured for each muscle. Of all the fibres examined only a veryfew were suitable for myofibril measurements. This was especially the case with the smallerfibres in which the myofibrils ran obliquely and did not exhibit a true cross-section (see Fig. 5).

Electron microscopy. The muscles used for electron-microscopic examination were all preparedin the following way. Mice of different ages were killed by dislocation of the cervical vertebrae,the forelimbs were skinned and the carcass pinned to cork board with the forelimbs in the fullyextended position. Glutaraldehyde fixative was then continually pipetted on to the bicepsbrachn muscles for 10—15 mm After this time the muscles were dissected from the limb andplaced in glutaraldehyde fixative for a further 1-75 h. The fixative used was 2-5 % glutaraldehydesolution buffered at pH 7-3 with 0 1 M phosphate buffer and containing glucose. After fixationthe muscles were washed overnight in o-i M phosphate buffer and post-fixed for 2 h in OsO4.The muscle fibres were then teased apart in 7 0 % ethanol, dehydrated in 100% ethanol andshort lengths of single fibres or small fibre bundles were embedded in Araldite (Ciba). Thesingle fibres were cut into 2 lengths and embedded in Araldite in the same plastic container(top from a plastic sample tube 2 cm in diameter) After the Araldite had hardened it was cut

Proliferation of myofibrils 595so that one of the fibre lengths could be sectioned transversely and the other longitudinally. Inselecting single fibres for embedding in this manner an attempt was made to select fibres ofdifferent sizes from the same muscle. Transverse and longitudinal sections of Araldite-embeddedfibres and fibre bundles were cut at a thickness of approximately ioo nm using an LKBUltrotome I. Ribbons of sections showing a gold interference colour were collected on carbon-or Celloidin-coated grids, and stained in lead citrate solutions. The sections were examined witha Siemens Elmiskop iA or a JEOL JEM 7 A electron microscope. In order to ascertain thecross-sectional area of each fibre, transverse sections were cut at 1 /im. These were stained in atoluidine blue/Best's carmine mixture and photographed under the light microscope. Thecross-sectional area of the fibre was measured using a planimeter.

RESULTSThe results of the measurement of myofibril number and size in fibres from mice of

different ages are given in Figs. 1 and 2. From Fig. 1 it will be seen that the numberof myofibrils within a fibre increases from about 75 in the fibres or myotubes of thenewborn animal to over 1000 in some of the fibres of the mature mouse. Thus myo-fibrils may proliferate within a single muscle fibre by as much as 10-15 times duringthe life-time of the animal.

It will be seen from Fig. 2 that the mean size of the myofibrils also increases, but to

1300

1200

1100

1000

900

0 800.0

Ec 700

1 600o

E 500

400

300

200

100

I I

250 500 750 1000 1250 1500 1750 2000 2250 2500

Fibre cross-sectional area I

Fig. 1. Graph showing the relationship between the number of myofibrils and the sizeand age of muscle fibres. The total number of myofibrils within individual muscle fibresis plotted against the fibre cross-sectional area. The fibres in which the myofibrils weremeasured were taken from mice of the following body weights and ages: # , 2 g (2 days),A, 10 g (2-5 weeks); O, 20 g (6 weeks); • , 30 g (6 months); D, 4° g (' year).

596 G. Goldspink

a lesser extent than the increase in number. As shown in Fig. 2 the mean myofibrildiameter shows maxima at certain fibre sizes; namely 150, 500, 1250 and 2600/tm2.The maxima tend to increase as the muscle matures. The maximum for the fibres ofthe newborn muscle is approximately 0-9 /im mean myofibril diameter whereas in themore mature muscles (30-g and 40-g mice) it is approximately i-o to I-I /mi.

1 3 r

1 2

I1'I 1 0

-6

t 08

| 0 7

06

050 250 500 750 1000 1250 1500 1750 2000 2250 2500

Fibre cross-sectional area (/trn2)

Fig. 2. Graph showing the relationship between the size of the myofibnls and the sizeand age of muscle fibres. The mean myofibril diameter for individual muscle fibres isplotted against the fibre cross-sectional area. The fibre in which the myofibrils weremeasured were taken from mice of the following body weights and ages: # , 2 g(3 days); A, 10 g (2-5 weeks); O, 20 g (6 weeks); • , 30 g (6 months), D, 40 g(1 year)

Figure 3 presents histograms of the distribution of myofibril diameters for themuscles of different ages. These histograms were constructed from a ioo-myofibrilsample obtained by measuring the diameter of 10 myofibrils in each of 10 differentfibres taken from the same muscle. All the histograms except perhaps the one for the30-g mouse are seen to be distinctly bimodal. In the younger mice (2 and 10 g) themyofibrils show size-distribution peaks at 0-5 and 0-9 /tm, whereas in the larger mice(20, 30 and 40 g) the second size peak occurs at the slightly higher value of i-o or1-i /tm. The maximum range of size appears to be 0-4—1-2 /im. In other words, themaximum myofibril size does not increase more than 3 times throughout the growthprocess.

Electron-microscopic examination of longitudinal sections of muscle fibres of dif-ferent sizes (see Table 1) revealed that in some fibres a high proportion of the myo-fibrils appeared to be in process of splitting longitudinally. All the fibres from thevery young muscles (differentiating fibres) contained split myofibrils and some of thefibres from the more mature muscles also contained a considerable number of splitmyofibrils. Examples of split myofibnls are shown in Figs. 6 and 7. From theseelectron micrographs it will be seen that the Z disks at one end of the sarcomere are

Proliferation of myofibrils 597•40

35

30

25l_Qi

_Q

E 20

z,15

10

5

0 5 0 751 0 0 5 0 751 Q 0 5 0 751 0 0 5 0 751 0 1 25

Myofibnl diameter C"m)

0 5 0 751 0 1 25

Fig. 3. Histograms of myofibnl sizes for the biceps brachii muscle from mice of dif-ferent ages Each histogram is constructed from 100-myofibril sample obtained bymeasuring 10 myofibnls in 10 different fibres The ages are, from left to right, 2 days,2-5 weeks, 6 weeks, 6 months, and 1 year, and the corresponding body weights are 2,10, 20, 30 and 40 g.

Table 1. The incidence of splitting of myofibrils as seen in longitudinal electron-microscope

sections. Fibre cross-sectional area was measured from transverse sections of the same

fibre.

Fibre no.

AxA,A,Bx

B,B3Dx

CxExE,Fxx,YxY2

Mouse, wt in g

40 (mature male)4 0

4°4 0

4 0

4 0

Cross-sectionalarea, /Jm!

137573O6 2 0

1215524

167S18 (half-grown male) 595181818182 (newborn)2 „2

57°560

11855 2 0

125

3369

Stage offibre growth

large phaseintermediatesmall phaselarge phasesmall phaselarge phasesmall phasesmall phasesmall phaselarge phasesmall phasemyotubemyotubemyotube

No. ofmyofibrilsin section

3642

3 i

2428

23

41

3516

192 1

1 0

11

6

No. ofmyofibnlssplitting

81

0

2

0

51

1

2

40

342

598 G. Goldspink

intact, while those on the other side are divided. It will also be noted that there areelements of the sarcotubular system in the fork of each split. The presence of thesarcotubular system and sometimes mitochondria between the ' daughter' myofibrilsprovides good evidence that the splits are not a fixation artefact or the result of themechanical handling of the tissue prior to fixation.

In each case the split myofibrils were examined over as long a length as possible;that is to say until the myofibril went out of the plane of the section or to the edge ofthe section. In all cases the 'daughter' myofibrils remained divided from each otherfrom the point of the split and as far as could be seen they never united with any

- 5

0 1 0 3 0 5 0 7 0 9 1 1 1 3 1 5 1 7

Fibril size (fm) Position of split (%)

Fig. 4. Histograms obtained by measuring the thickness at the A-band level of 25 myo-fibrils which were in process of splitting (horizontal hatch) and 25 which were not split-ting (vertical hatch). It will be seen that the distribution peak for the splitting myofibrilsis at approximately twice the values for the non-splittingmyofibnls. Also, it will be notedthat the extent of the overlap (shown as the cross hatch) of the size distributions of thesplitting and non-splitting myofibnls was not very great, considering these measure-ments were taken from longitudinal sections. Given on the right are the histogramsobtained by measurement of the position of the splits with respect to the longitudinalaxis of the myofibrils (solid columns). The position of the split was measured by com-paring the size of the daughter myofibrils with the unspht part of the parent myofibriland expressed as a percentage.

Proliferation of myofibrils 5 99

adjacent myofibrils. In a very few cases when a myofibril had split more than once, withthe splits occurring at slightly different transverse levels, they gave the impressionthat the ' daughter' myofibrils had coalesced with adjacent myofibrils; closer examina-tion revealed that these were most probably not true myofibril anastomoses butmultiple asymmetric splits.

In order to obtain information about the size at which myofibrils normally split, theA-band depth (maximum myofibril diameter in section) in 25 splitting myofibrils and25 myofibrils which were apparently not splitting was measured from longitudinalsections taken from both differentiating fibres and fibres undergoing further growth(hypertrophy). The sampling procedure used was to measure the 25 splitting myo-fibrils in the order that they were found and to measure 25 non-splitting myofibrilsadjacent to the splitting myofibrils from the same photographic plates. Ideally thesemeasurements should have been made from transverse sections but this would havenecessitated the preparation of serial sections for electron microscopy through atissue depth of several sarcomeres in order to detect a split (ribbons of 200 to 300serial sections). This, and the fact that there was only a remote chance of a splitoccurring at the level from which the sections were taken, made the measurement ofsplitting myofibrils from transverse sections impracticable.

The results of the measurements of the splitting and non-splitting myofibrils arepresented as frequency histograms in Fig. 4. Also given in Fig. 4 in the same form aremeasurements of the position of the splits with reference to the myofibril axis. Thesewere measured as the percentage size of each of the 'daughter' myofibrils as comparedwith original' parent' part of the myofibril. The modes of the histograms for the split-ting and non-splitting myofibrils clearly indicate that the splitting myofibrils wereabout twice the size of the non-splitting myofibrils. There was, however, an interest-ing difference between the young, differentiating fibres and the older fibres whichwere undergoing hypertrophy. The histograms indicated that the myofibrils of theyoung fibres split when they reach a diameter of about 0-8—0-9 / t m ' whereas thosein the older fibres apparently split when they attain a diameter of approximately i-o-I-I fim. The results of the measurements of the position of the split indicated that themyofibrils usually split approximately down the middle in both differentiating fibresand fibres undergoing further growth.

DISCUSSION

As indicated in the Introduction, the study of the structural changes that occurduring muscle growth is made difficult by the fact that the constituent fibres of amuscle do not grow synchronously. In order to obtain muscle fibres that are producingmyofibrils during the late differentiating process it is only necessary to use musclefrom very young mice (newborn to 1 week old). The fibre hypertrophy process how-ever has proved to be a much more elusive process to study, as at any one time onlya small percentage of fibres (less than 10%) is undergoing hypertrophy. However, theuse of phase-contrast microscopy for the measurement of the myofibrils in fibres ofdifferent ages and sizes provided some very useful information about post-natal fibregrowth.

6oo G. Goldspink

The bimodal distributions of the myofibril size measurements shown in Fig. 3strongly suggested that myofibril proliferation during muscle fibre growth involvesthe longitudinal splitting of the already existing myofibrils. In the mouse it appearsthat the myofibrils in fibres which undergo the most extensive growth split four timesduring the animal's lifetime. Thus the number of myofibrils increases from 75 initiallyto about 1200. In animals with larger muscle fibres it is probable that the process ofbeing built up in size and then splitting is repeated more than four times. In the mousethe process of muscle'fibre growth has been shown to take place in a stepwise fashion,most of the fibres occurring at 100, 375 to 500 and 1250 fim2, depending on the muscleand the age of the animal (Goldspink, 1962; Goldspink & Rowe, 1968; Rowe & Gold-spink, 1969). Also in some muscles, for example, the sternomastoideus of the mouseand the biceps brachii of the rat, a fibre size distribution peak is also apparent at about2600 /tm2. It is not quite certain why these sizes or levels of growth are, as it were, thestable levels; however, they do correspond to the peaks for the mean myofibril sizeas shown in Fig. 2. In other words, the size of the fibre is most stable when the myo-fibrils are just below the size at which they split. A search of the literature revealedthat myofibril splitting had previously been recorded by Heidenhain as early as 1913in differentiating trout muscle.

In spite of the fact that the measurements of splitting and non-splitting myofibrilswere made from longitudinal electron-microscope sections, the histograms clearlyindicated that the splitting myofibrils were about twice the size of the non-splittingmyofibrils. The finding that the myofibrils usually split approximately down themiddle when they attain a certain size presumably explains why the myofibrils are allmore or less the same size within the same fibre, but not in the same muscle.

There are also several other features of this splitting mechanism that fit the knownfacts. For instance, the cross-striations of new myofibrils formed in this way will havethe same register and sarcomere length as the existing myofibrils. There is some evidencethat the production of the first myofibrils during early myogenesis takes place bythe assembling of free actin and myosin filaments into the myofilamental array byinteraction of the actin filaments with the cross-bridges on the myosin filaments(Fischman, 1967). Certainly the sarcomere length in very young muscles (2-2-2-3/tmat maximum limb extension) is the length at which there would be maximum inter-action between the actin filaments and the cross-bridges on the myosin filaments.As the muscle fibres lengthen during growth the sarcomere length increases to about2-8 /an maximum limb extension (Goldspink, 1968). Therefore, if the muscle fibrescontinued to produce myofibrils using the original method of assembly, the new myo-fibrils would be expected to have a considerably shorter sarcomere length than theexisting myofibrils. This cannot be the case as there are usually no wide discrepanciesin the sarcomere length in the same fibre, or even in the same muscle. No evidence ofde novo synthesis of myofibrils was seen in any of the fibres examined. Also, no verysmall myofibrils were seen as would be expected if de novo synthesis were the methodof proliferation.

It is of interest to speculate on the mechanical reasons for the splitting of the myo-fibrils when they attain a certain size. Presumably, there must be a strain created

Proliferation of myofibrils 601

within the myofibril as it grows so that when it reaches a critical size the Z disks rip,causing the myofibril to split longitudinally. The first possible explanation for thisis based on the observation from electron micrographs that the A band invariablyappears to have a greater diameter than the I band. Indeed, recent work has shownthat the distance between the A filaments is variable and depends on the sarcomerelength and ionic environment of the myofibril (Brandt, Lopez, Reuben & Grandest,1967). Thus it appears that there must be a discrepancy between the spacing of theA filaments and the spacing of the I filaments. Certainly a discrepancy would be ex-pected, due to the fact that the A-filament lattice is hexagonal (Huxley, 1957) and theI-filament lattice at the level of the Z disk is square or slightly rhombic (Reedy, 1964;Carlsen & Knappeis, 1963). If the width of the A band is greater than the I band theperipheral I filaments would be pulled at an angle slightly oblique to the myofibrilaxes. This oblique pull would cause a strain at the centre of the Z disks and this wouldbe exaggerated when the A filaments move further apart with the shortening of thesarcomeres. Under these conditions the Z disk would be expected to rip when themyofibrils attained a certain size, because as the myofibrils increase in girth duringthe growth of the fibre the sarcomeres will increase in strength (as there will be morecross-bridges in parallel). Hence the strain on the centre of the Z disks would besufficient to cause them to rip.

Alternative explanations are that there may be a tendency for uneven activationof the myofibrils when they attain a certain size. This then may result in some halfsarcomeres contracting momentarily before the rest of the sarcomeres, thus causingthe Z disks to shear. Also it is possible that the splitting of the myofibrils is due to amajor disruption of the filament lattice by the encroachment of the sarcoplasmicreticulum.

It is difficult to obtain direct evidence that myofibrils split by either or both ofthese processes. However, some evidence that tends to support the first explanationis that frequently elements of the sarcotubular system are seen in apparent isolationin the centre of the myofibrils of differentiating fibres. These disruptions of the latticeare quite often seen in transverse sections of myofibrils in almost the exact centre ofthe A band. In some cases the disruption of the lattice is seen to extend from thecentre to one edge of the myofibril. It has been shown by Reedy (1964) that the Z diskhas a weave-like appearance and it is quite attractive to visualize the Z disk ripping likea piece of cloth. That is to say, when a few of the central Z-disk filaments are brokenthen the rip would extend to the edges with the direction of the' weave'. This would ofcourse result in quite a straight break in the Z disk, and this may well be why myofibrilsin adult muscles tend to be straight-sided; i.e. square or rhombic in shape ratherthan ovoid or spherical when examined with the electron microscope.

Further work is now in progress aimed at the elucidation of the precise mechanismof myofibril splitting and the ultrastructural changes that take place during fibregrowth. Particular attention is being given to the study of any changes that may occurin the lattice of the Z disk during growth and during contraction.

602 G. Goldspink

The author wishes to acknowledge the expert technical assistance rendered by Miss JaniceHaider; Mr J. Fotheringham and Mr S. Waterson. While this work was being carried outthe author was in receipt of Research Grants from the Agricultural Research Council and theMuscular Dystrophy Group of Great Britain.

REFERENCES

BRANDT, P., LOPEZ, E., REUBEN, J. & GRUNDEST, H. (1967). The relationship between myo-filament packing density and sarcomere length in frog striated muscle J. Cell Btol. 33, 255-263.

CARLSEN, F. & KNAPPEIS, G. G. (1963) Further investigations of the ultrastructure of theZ disc in skeletal muscle. Acta phystol scand. 59, 213-215

FISCHMAN, D. A. (1967). An electron microscope study of myofibnl formation in embryonicchick skeletal muscle. J. Cell Bwl. 32, 557-575.

GOLSDPINK, G. (1962). Studies on post-embryonic growth and development of skeletal muscle.1 Evidence of two phases in which striated muscle fibres are able to exist. Proc. R. Ir. Acad.62, B id, 135-150.

GOLSDPINK, G. (1964). The combined effects of exercise and reduced food intake on skeletalmuscle fibres J. cell. comp. Phynol. 63, 209-216.

GOLDSPINK, G. (1965). Cytological basis of decrease in muscle strength during starvation. Am.J. Physiol. 209, 100-114.

GOLDSPINK, G. (1968). Sarcomere length during the post-natal growth of mammalian musclefibres. J. Cell Set. 3, 539-548.

GOLDSPINK, G. & ROWE, R. W. D., (1968). The growth and development of muscle fibres innormal and dystrophic mice. In Research in Muscular Dystrophy, Proceedings of the 4thSymposium, pp. 116—131. London: Pitman Medical Publishing

HEIDENHAIN, M. (1913). Uber die Entstehung der quergestreiften Muskelsubstanz bei derForelle. Arch mikrosk. Anat. EntivMech 83, 427-522.

HUXLEY, H. E. (1957). The double array of filaments in cross-striated muscle. J. btophys. bio-chem. Cytol. 3, 631-648.

REEDY, M. K. (1964). Remarks at a discussion on the physical and chemical basis of muscularcontraction. Proc. R. Soc. B 160, 458.

ROWE, R. W. D. & GOLDSPINK, G. (1968). Surgically induced muscle fibre hypertrophy. Anat.Rev 161, 69—76.

ROWE, R. W. D. & GOLDSPINK, G. (1969). Muscle fibre growth in five different muscles inboth sexes of mice. J. Anat. 104, 519-530.

STEEDMAN, H F. (i960). Section Cutting in Microscopy. Oxford: Blackwell.

{Received 26 June 1969)

Fig. 5. Phase-contrast photomicrograph of a transverse section of muscle fibres in a20-g, 6-week-old mouse. Note the difference in size and arrangement of the myofibnlsin the fibres of different sizes, x 2000.

Figs 6, 7. Two electron micrographs of splitting myofibnls. Note that the Z disk atone end of the sarcomere is intact, whilst that at the other is split into two, with ele-ments of the sarcotubular system occurring in the fork of the split, x 25 000.

Proliferation of myofibrils 603

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