The onset of myogenesis in denervated mouse skeletal muscle regenerating after injury

Neurcwciewe Vol. 28, No. 2, pp. 509-514, Printed in Great Britain

03~522/89 $3.00 + 0.00 Pergamon Press pk 0 1989 IBRO

1989

THE ONSET OF MYOGENESIS IN DENERVATED MOUSE SKELETAL MUSCLE REGENERATING AFTER INJURY Departments

J. K. MCGEACHIE and M. D. GROUNDS* University of Western Australia,

ofAnatomy and HumanNedlands, Biology, and *Pathology, 6009 Australia

Abstract-Denervation of skeletal muscle stimulates increased turnover of muscle nuclei and connective tissue cells. The present inv~ti~tion tests whether denervation “primes” myogenic precursor cetls, so that the onset of DNA synthesis in muscle precursors after traumatic injury occurs earlier than in innervated muscle. The left legs of 29 male BALBc mice were denervated, and 1 week later small incisions were made in the tibialis anterior muscles of both legs (denervated and innervated). At specific times after injury each mouse was injected once with tritiated thymidine to label replicating muscle precursors. Muscle lesions were sampled 10 days after injury (when all precursors had fused to form myotubes) and prepared for autoradiography. The presence of labelled myotube nuclei showed that muscle precursors had been synthesizing DNA at the time when [~Hlthymidine had been injected. Our data suggest that very few precursors were proliferating in denervated muscle within 30 h after injury, and the onset of myogenesis at 30 h was essentially the same in denervated and innervated muscle. The retrospective analysis indicates that there were also similar proportions of muscle precursors proliferating at different times after injury, in regenerating lesions of both denervated and innervated muscle. Thus, denervation does not stimulate an earlier regenerative response in injured skeletal muscle.

When skeletal muscle is denervated fibre atrophy is conspicuous. In addition, there are ultrastructural changesl”rs and biochemical changes in muscle nuclei

with respect to histone composition” and phosphorylation of nuclear proteins.12 Although the nuclei of denervated muscle are affected, it is not known whether they become “primed”, as was proposed by Studitsky et al.,” who considered that muscle regeneration was improved in grafts of denervated muscle due to increased muscle “plasticity”. A number of other studies have shown that following injury or mincing of denervated muscle, the early myoblastic stages of the regenerative response were accelerated (reviewed by Carlson’). Denervation does appear to increase the early regenerative potential of small free whole muscle graftq4 although more recent studies indicate that pre-denervation does not contribute to the long term success of small muscle transplants6,rq In the present paper we test the possibility that denervation “primes” (or activates) the nuclei of muscle precursors in mature skeletal muscle. It is considered that these muscle precursors are the satellite cells which lie between the sarcolemma and basal lamina of muscle fibres,2 and there is ample evidence that a substantial increase in numbers of satellite cells occurs in denervated skeletal muscle.‘3,23*24,27 Moreover, this increase is associated with increased prohferation and turnover of muscle and connective tissue nuclei.‘“‘92’z We compare the onset of DNA synthesis in muscle precursors in denervated and innervated muscle of adult BALBc mice regenerating after a simple cut injury, using an autoradiographic model of myogenesis in viva. Previous studies with this model show that the onset of DNA synthesis in muscle precursors

of innervated BALBc mouse injury.m If denervation does we hypothesize that muscle activated earlier than 30 h in denervated muscle. EXPERIMENTAL The

muscle is at 30 h after “prime” satellite cells, precursors would be regenerating lesions of

PROCEDURES

right legs of 29 inbred male BALBc mice (from the Animal Resources Centre, Murdoch University, Western Australia) aged &g weeks were denervated by removing 5 mm of sciatic nerve high in the thigh. Seven days after denervation the tibialis anterior muscles of the right (denervated) and left (control innervated) legs were injured by a delicate cut inflicted with a razor (described in detail by McGeachie and Groundszc). To label replicating myogenic cells, tritiated thymidine (specific activity 5Ci/mmol; Amersham, England) was injected intraperitoneally at a dosage of 1 &i/g body weight at times ranging from 12 to 72 h after injury (Table 1). Bach mouse received only one injection of r3H]~ymidine. At 10 days after injury mice were anaesthetized with ether and killed by cervical dislocation. The rationale for this experimental regime was based on the fact that when [3H]thymidine is injected, it is available to replicating cells for about 30mins [3H]Tbymidine, which is not incorporated into DNA during this time, is metabolized and excreted. Cells synthesizing DNA in preparation for mitosis at the time of [3H]thymidine injection, incorporate it into their nuclei and pass the label on to subsequent daughter cells: this label becomes progressively diluted with each cell division. Muscle precursor cells which incorporate [‘Hlthymidine carry the label through to myoblast and myotube nuclei. Therefore, the presence of labelied myotube nuclei in regenerated muscle 10 days after injury indicates that muscle precursor cells had been synthesizing DNA at the time of [rH]thymidine injection. Thus, a retrospective analysis of the initiation and duration of muscle precursor replication can be achieved. All samples were fixed in 10% formaldehyde, post-fixed in 1% osmium tetroxide, block-stained with p-phenylenediamine, embedded in Araldite and 1pm sections processed for 509

510

Table

J. K. 1. Proportions

Time after injury of [?H]thymidine injection (h) 12

of myotube denervated

18 20

and M. D. GKWN~S

nuclei labelled, and distribution of grain counts, in muscle samples and innervated tibialis anterior muscle 10 days after cut injury Denervated 13-24

Grains/nucleus: % Labelled myotube nuclei

(3) (3)

MCGEACHIE

25-50

(100) (100)

(IFi?) (1W (1:) 20

24 26 30

36

42 48 54 60 12

(35) (3) (2) (2) 6 (:) (1) 8 7 18 5 17 25 20 13 21 21 19 35 26 35 31

Mice were injected with [)H]thymidine

from

Innervated 7-12 13~-24 25 SO

% Labelled myotube nuclei

17

(F) (2)

34

removed

(20) (75) (100) 88 (Ii) (100) 60 83 86 89 76 52 63 81 48 31 24 18 I1 51 42 (1 pa/g)

(60) (25)

(20)

22 9

36 17 11 11 18 36 27 19 31 32 65 32 14 28 34

(4) (4) (2) (2) (1) (2) (1) (3)

(100) (1W (100) Cl@3 (100) (1W (100) (1W

(40)

(100)

(46) (1) 9 7 9 16 I 29 29 20 39 38 16 46 36 19 40 29

(1::) (100) 12 73 71 87 92 72 37 74 35 53 53 31 42 63 53 31

.~ -

7

4 3 6 12 10 21 31 11 44 50 12 21

6 25 3 3

at O-72 h after injury (where < 5% of myotube

22 27 29 13 8 26 51 21 44 23 41 19 33 24 33 32

6

2 IO 5 18 20 6 31 15 5 13 34

2 3 4 13 10 8 1 3

nuclei were labelled the data

are shown in parentheses).

autoradiography, as described by McGeachie and Grounds.20 For the analysis, about 200 myotube nuclei in each of the 29 denervated and innervated muscle lesions sampled 10 days after injury were examined, and the numbers of labelled myotube nuclei and grain counts per nucleus were counted. RESULTS

Histology Muscle regeneration in cut lesions examined 10 days after injury was similar in denervated and innervated (control) muscle: densely packed myotubes with central nuclei were present with little intervening fibrous connective tissue (Fig. 1). Areas of uninjured denervated muscle adjacent to the lesion showed typical atrophy (Fig. 1). Labelling of myotube nuclei in lo-day-old lesions There was minimal (< 5%), low (3-5 grains) labelling of myotube nuclei in all lesions in innervated muscle where [‘Hlthymidine was injected prior to 30 h after injury (Table 1). Previous autoradiographic studies indicate that low labelling (3-5 grains) at this time is due to reutilization of [)H]thymidine. However, in denervated muscle injected prior to 30 h after injury, some myotube nuclei were labelled with more

than 5 grains per nucleus; high grain counts in myotube nuclei at 18 h were 8 and 13; at 20 h 7 and 10; at 24 h 6, 7, 7, 9 and 15, and at 26 h 8 (see Discussion). In mice injected with [‘Hlthymidine at 30 h or more after injury, more myotube nuclei were labelled and grain counts were higher in both denervated and innervated muscles (Table 1). Autoradiographic grains are clearly shown in myotube nuclei in Fig. 2. There was no difference between denervated and innervated muscle in the time when substantial muscle precursor replication first started (3@36 h). There was little difference in the proportions of labelled myotube nuclei in denervated and innervated muscle, particularly in samples from mice injected up to 36 h after injury (Table 1 and Fig. 3). Although there was an apparent decrease in proportions of labelled myotube nuclei when [3H]thymidine had been injected at 4260 h (Fig. 3) in denervated muscle (compared with innervated), this could easily be accounted for by biological variation between animals, as wide variation is evident in duplicate samples of innervated muscles at these times. As there were only two data points for each time period for denervated and innervated muscle between 42 and 60 h, the data were not amenable to statistical analysis.

Myogenesis in regenerating denervated muscle

Fig. 1. Muscle regeneration 10 days after a minor cut injury to (A) denervated and (B) innervated (control) muscle. Note the atrophy of intact denervated muscle fibres adjacent to the lesion in (A), and the prevalance of myotubes in both lesions.

DISCUSSION

In lesions of both denervated and innervated muscle, the onset of myogenesis at 30 h, and the low proportion of myotube nuclei labelled (10 days after injury) when [‘Hlthymidine had been injected up to 42 h after injury, is similar to previous experiments using BALBc mice. ” Autoradiographic studies of regenerating innervated muscle of adult chickens’ also indicate that few muscle precursors are replicating before 30 h after injury. However, in innervated

muscles of adult male and female Swiss mice the onset of myogenesis is at 24, rather than 30 h after injury.‘O When EDL muscles in BALBc mice are autotransplanted the initiation of myogenesis occurs somewhat later, at 48 h, due to diffusion barriers between the host and transplanted muscle, and the lack of revascularization of the transplant.25 In the present experiment, in samples injected up to 72 h after injury there was no difference between the proportions of labelled myotube nuclei in regenerated lesions of denervated and innervated muscle. Our data suggest

J. K. MCGEACHUZ and M. D. GROUNDS

Fig. 2. Autoradiographic labelling of myotube nuclei in a sample of regenerating denervated muscle where muscle precursors were labelled with [‘Hlthymidine injected at 54 h after injury. (A) Autoradiographic grains in focus. (B) The same field with the underlying tissue in focus.

that the response of satellite cells to injury is similar in denervated and innervated muscle. The results do not support the original hypothesis that denervation “primes” satellite cells and stimulates the early initiation of myogenesis in denervated muscle after injury. The small proportion (less than 6%) of highly labelled (6-15 grains) myotube nuclei seen in denervated muscle which had been injected with [‘Hlthymidine before the onset of myogenesis (30 h), can be accounted for by the labelling of a small number of satellite cells activated by denervation, and their subse-

quent incorporation into myotuhes. The proliferation of satellite cells in response to denervation is well documented.” ‘9.2’,22Within 2 days after denervation 10% of satellite cells are proliferating and they have been calculated to reach levels of “14-40%” by 3-4 days. 2’ These derived values appear high, since it has been shown in outbred Swiss mice injected once daily with [3H]thymidine over the first 7 days after denervation, that 11% of muscle nuclei and 41% of connective tissue nuclei become accumulatively labelled. I6 In BALBc mice (from the same inbred colony as used

513

Myogenesis in regenerating denervated muscle 60 -

p

o-- - - - --c

Innervated muscle

-

Denervated muscle

0

40-

: d g

30.

E” H $

20.

6 * 10 -

OI

.

8

12

18

24

30

Time of 13Hl thymidine

38

42

injection

I

I

48

84

after injury

80

72

(Hours)

Fig. 3. Graphical representation of data from Table 1, showing the proportions of labelled myotube nuclei in muscle lesions 10 days after cut injury to denervated and innervated muscle. [‘HJThymidine was injected at the times shown on the horizontal axis: each mouse received only one injection of r3H]thymidine. The lines which join the mean percentages of labelled myotube nuclei suggest an apparent difference between proportions of myotube nuclei labelled in denervated and innervated muscle, when [“Hlthymidine had been

injected from 42 to 60 h after injury. This can probably be accounted for by biological variation between samples, which is particularly pronounced in samples where large numbers of muscle precursors were replicating.

in the present experiment) injected once daily with [‘H]thymidine during the tist 7 days after denervation, there is an accumulated labelling level of 3.5% of muscle nuclei.” This level of turnover is maintained over the first 4 weeks after denervation. By comparison, innervated control muscles in the same mice have consistent labelling levels of 0.1% of muscle nuclei (accumulatively over 7 days).” The calculated daily turnover of muscle nuclei in denervated muscle would be OS%, compared with only 0.014% in innervated muscles. Therefore, in the present experiment, a single injection of [‘Hlthymidine at 7-8 days after denervation, i.e. within 30 h of injury, should label less than 1% of these proliferating satellite cells. It seems likely that these labelled proliferating satellite cells in denervated muscle would be incorporated into myotubes formed after injury, and would result in about 2-4% of myotube nuclei being significantly labelled, if the labelled satellite cells divided only 2-3 times before fusing into myotubes. This number of muscle precursor divisions before fusion is known to occur in BALBc mouse muscle regenerating after injury.’ One problem inherent in [3H]thymidine labelling experiments is the variable uptake of [‘Hlthymidine due to cell cycles not being synchronized, and to differences in DNA synthesis during the S phase of the cell cycle.’ This means that autoradiographic grain counts in post-mitotic myotube nuclei could be either the result of an initial low uptake of [‘Hlthymidine

at the time of injection, or alternatively be due to progressive dilution of an initially high label with a number of mitotic divisions. The authors are mindful of such problems with these experiments.’ Nevertheless, the grain counts of post-mitotic myotube nuclei 10 days after injury clearly show the onset of DNA synthesis in muscle precursors, and also give some indication of the relative proportions of precursors proliferating in denervated and innervated muscle at different times after injury. It appears that about 30 h may represent a minimum time for quiescent muscle precursors of BALBc mice to become activated after injury, and it is not known whether this time interval can be shortened experimentally. Similar autoradiographic studies in chickens’ and Swiss mice,” indicate that a minimum of 24 h must elapse after injury before quiescent muscle precursors start to synthesize DNA. This time interval may be slightly shorter in rats, since it has been shown that satellite cells of growing rats start to synthesize DNA at 20 h after injury,26 and observations in tissue culture also imply that the onset of DNA synthesis in satellite cells of adult rat muscle is around 20 h (i.e. 2-4 h before mitosis, reported at 24 h).’ CONCLUSION

Although denervation of skeletal muscle causes an increase in satellite cells and connective tissue cell

J.

514

K. MCGEACHIE and

turnover. it does not “prime” the general population of muscle precursors to start synthesizing DNA more rapidly after injury than in innervated muscle. Moreover, denervation does not appear to enhance the process of muscle regeneration compared with innervated muscle, as indicated by the proportions of labelled myotube nuclei, and hence muscle precursors proliferating, at different times after injury.

M. D. GK~UNDS

Ac,knorr,led~ements-The authors gratefully acknowledge the technical assistance given by Mr S. Parkinson, Mr A. Cockson, Mr M. Holmes, Mr A. Minton and Mr M. Thompson, the typing by Mrs M. Seats, and the photography by the Department of Medical Illustration. This research was supported by the Nicholas and Eliza McCluskey Memorial Bequest to Dr John McGeachie, and a grant from the National Health and Medical Research Council of Australia to Dr Miranda Grounds.

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