edna-muscle.html

ENHANCED MUSCLE REGENERATION AND INCREASED STRENGTH IN MYOPATHIC ANIMALS TREATED WITH ETHYLENEDINITRAMINE (EDNA)

PAUL PIETSCH and JOHN HARROLD

Biochemical Research Laboratory, The Dow Chemical Company, Midland, Michigan*

Adapted from an article originally published in Nature 213:601-3, 1967
Web Contact: pietsch@indiana.edu

Abstract

Myogenesis is triggered by cellular events associated with growth. The high-impact explosive, ethylenedinitramine (EDNA) enhances growth in plants. Would EDNA, in turn, enhance regeneration in mammalian skeletal muscle?

In mechanically wounded mouse muscle, EDNA treatment dramatically increased both the histological and the biochemical signs of regeneration.
In mice with genetic muscular dystrophy, EDNA treatment dramatically increased regeneration. Prolonged treatment neither cured the animals, nor increased their body weight, but they did exhibit improved ability to climb and use previously immobile hind limbs.
Treatment with EDNA enhanced the strength of dogs with dietetically induced myopathy.

DIFFERENTIATION of skeletal muscle is activated as a function of replication (1). This predicts that enhancement of growth in a myogenic system will increase the yield of new muscle. Excessive proliferation, however, can cancel muscle differentiation (2). In employing the theory it is therefore necessary to stipulate that increased growth shall not reach deleterious levels (although just what theses levels are cannot yet be specified). The properties of ethylenedinitramine (EDNA){3, 4} seemed, intuitively, to satisfy this condition.**

Wounded muscle (mouse)

EDNA was converted to its ammonium salt and dissolved in physiological saline. Young adult Swiss-Webster mice (for description see ref. 5) which had been wounded in their tibialis anterior muscles 45 h earlier received 0.1 mg of EDNA by intraperitoneal injection. Wounds were fixed 2 days later (96 h after wounding) and were examined histologically. Those from treated animals showed more regeneration than controls by a factor of almost four (Table 1). The effects of EDNA were much less pronounced when the compound was applied before or 24 h after wounding (Table 1).

EDNA did not stimulate grossly premature unfolding of the signs of differentiation of new muscle. This was judged from the results obtained in the following experiment: three mice with 45 h old wounds were given EDNA as before; the tissues, together with those of three untreated controls, were fixed at 84 h; that is, about 10 h before the advent of the differentiating muscle fibers known as myotubes. (In regenerating mouse muscle, myotubes first appear in small numbers at 94.5 h after wounding. Within an h the density of myotubes increases almost ten-fold and tends to hold steady until early in the sixth day when there is another increase in myotube numbers. These are unpublished observations of Pietsch and McCollister.) Serial examination of 84 h old sections revealed not a single myotube among either the controls or the EDNA-treated specimens.

Biochemistry

In another series the tibialis anterior muscles of male C57BL/6J mice with the same birth date were punctured aspectically. Animals were divided into two equal groups. At 67 h those of one group received 0.1 mg of EDNA intraperitoneally. The others were untreated. All animals were given the equivalence of 10 uc of L-lysine labeled with carbon-14 (uniformly labeled with 200 mc./mmole) intraperitoneally 117 h (+/- 3 min) after wounding. Animals were killed 161 h after wounding, and their tibialis anterior muscles were dissected for the extraction of actomyosin. Synthesis of this protein complex was assessed from its radioactivity as determined by liquid scintillation counting. Specific activities, recorded in Table 2, were of the order of about 30-80 percent greater in EDNA-treated animals than among controls.

Dystrophic muscle (mouse)

The aforementioned evidence encouraged experiments with genetically dystrophic mice (Re 129/dydy). Muscle regeneration is spontaneous in these animals. Male dydy mice born within 3 days of each other were divided into two groups of three animals each. One group was maintained on ground 'Purina' chow and the other on the food taken from the same bag and blended with 0.2 % (w/w) EDNA. The tibialis anterior muscles of these animals were prepared for histological examination 13 days later. There were appreciably greater numbers of myotubes in the muscle of the three EDNA-treated animals than in their controls. To specify this in quantitative terms, eight sections (approximately 10 % of the muscle) were randomly selected from the tibialis anterior muscle of a control and an experimental animal that happened to have been litter mates. The number of myotubes estimated for each unit area showed that the factor of increase in regeneration was approximately two (Table 3).

During normal regeneration myotube nuclei increase progressively (7, 8). The number of nuclei in each tube reflect maturation and therefore serve as an index of the quality of regenerative activity. Tubes with smaller numbers signal the advent of new foci of regeneration. Tubes with greater numbers indicate more advanced stages of differentiation. In control dystrophic specimens the number of nuclei in a myotube tended to cluster between four and ten, and in no control case were there more than twenty nuclei in a tube. In EDNA-treated dystrophic muscle the number of nuclei in each tube varied considerably; there were many instances in which the number exceeded twenty (see Table 4 for more rigorous examination of this point).

EDNA-treatment improves climbing ability of dystrophic mice

Dystrophic mice maintained on control or experimental regimens over a period of several months showed no differences in survival rates or body weight. Muscular dystrophy was in no sense "cured"; after about 8 consecutive weeks, however, EDNA-treated animals had gained feeble, although measurable use of their hind limbs (Table 5). Slow motion films of animals taken before treatment and after several months of feeding on EDNA revealed a pumping motion in the hind limbs that was not observable at the outset of the investigation nor in control animals (see footnote to Table 5 .

Dogs with diet-induced myopathy

Effects of EDNA were evaluated in purebred male beagles. The dogs suffered a low-grade myopathy that had been induce by prolonged vitamin E deficiency and copper and selenium deprivation (9-11). There were eight dogs, four from each of two litters born 4 days apart. Animals were brought into the laboratory as weanling pups. They were immediately started on the vitamin E-deficient regimen with deionized water for drinking. A salt mixture free of copper and selenium was added to their diet along with vitamins other than E (refs. 9-11). One dog from each litter was kept on a normal kennel ration throughout the study. The deficient regimen was assayed with weanling rabbits (because of high sensitivity to vitamin E deficiency). Rabbits succumbed in 29 weeks. Estimating from the data of others (12), our methods were roughly 40 % efficient in producing muscle lesions.

Tests for the dogs

Dogs were trained for 9 months. Each animal was taught to pull a sledge counterpoised with his own body weight. The test course was 48 m, linear, smooth-surfaced, air conditioned and in a pressure-regulated room immediately adjacent to the living quarters. Each animal ably and eagerly negotiated the 48 m, reproducing his performances within a faction of a second. Weights could be varied +/- 10% without altering performance. Preliminary tests were conducted until there was no doubt that deficiently fed dogs went through the course more slowly than their normally fed siblings.

In the test itself, each dog was clocked through the course at roughly the same time of day, twice weekly for 42 weeks. The test interval was subdivided in four experimental periods (I-IV). Each dog's performance was analysed individually on the basis of differences between periods. Comparisons between dogs were made on the basis of differential (relative) rather than absolute values. No EDNA was administered to any dog during period I. During period II three deficient dogs were maintained on a daily dose of 10 mg/kg of EDNA fed by capsule; the other three deficient animals were untreated. During period III EDNA was withheld. In period IV two deficient but previously untreated dogs were given the compound along with one dog that had been treated during period II. Dogs, of course, were appropriately isolated at all times.

How well did the dogs perform?

Data compiled in Table 6 and analysed in Table 7 leave little doubt that EDNA acted in some manner to enhance pulling capacity. About 4 consecutive weeks of treatment with the compound were required before beneficial effects were manifested. About a week after withdrawal of EDNA the previously treated dogs showed marked increases in time through the course; that is, they became measurably weaker after the treatment was discontinued. Reintroduction of EDNA led to significant increases in speed, again after about 4 weeks.

References

01Pietsch, P. and McCollister, S. B., Nature, 208, 1170 (1965).
02Stockdale, F. and Holtzer, H. Science, 146, 866 (1964).
03Mees, G. C., J. Exp. Bot., 16, 48 (1963).
04Holmsen, T. W., Plant Physiol. suppl., 41, xlv (1966).
05Handbook of Genetically Standardized JAX Mice (Bar Harbor Times; Bar Harbor, Maine, 1962). 06
Lash, J., et al., Anat. Rec., 128, 697 (1957).
07Pietsch, P. Anat. Rec., 139, 167 (1961).
08Pietsch, P., Nature 203, 117 (1964).
09Hubbel, C. J., J. Nutrit., 14, 273 (1937).
10Mason, K. E., and Harris, R. S., J. Biol. Symposium, 12, 495 (1957).
11Mason, K. E., and Harris, R. S., Diets Manual, 5 (Nutritional Biochemical Company, 1964).
12Mason, K. E., in Structure and Function of Muscle (edit. by Bourne, G. H.), 3 (Academic Press, New York and London, 1960).

**Table 1.** EFFECTS OF EDNA ON REGENERATION IN MUSCLE AFTER SURGICAL INJURY
Experiment	Cases	Muscle fibers (regeneration/injured)	Index
Untreated	5	0.442+/-0.076 S.D.	100.00
EDNA, 24 h before wounding	4	0.685+/-0.077 S.D.	154.97
EDNA, 24 h after wounding	3	0.662+/-0.045 S.D.	149.77
EDNA, 45 h after wounding	5	1.759+/-0.220 S.D.	397.96
The above specimens were fixed and processed 96 h after wounding for reasons give in refs 6, 7 and 8. The rationale in the timing of experiments can be found in ref. 1.Three sections, about 10 % of the wound area, were randomly selected for counting in each case. S.D. is standard deviation.

**Table 2.**INFLUENCE OF EDNA ON THE SYNTHESIS OF THE CONTRACTILE PROTEIN ACTOMYOSIN IN REGENERATING MOUSE MUSCLE
ANIMALS	SPECIFIC ACTIVITY*
ANIMALS	Aliquot 1	Aliquot 2	Means
CONTROLS:			5,090
1	5,487	5,318
2	4,660	4,762
3	5,164	5,160
EDNA-treated:			7,997
1	6,325	6,206
2	8,564	8,765
3	8,818	9,009
*[DPM 14-C/OD actomysin x OD sarcoplasmic proteins] minus [specific activity of actomyosin from uninjured tibialis anterior muscles], where DPM is disintegrations per minute and OD is optic density at 275 nm.

**Table 3**.INFLUENCE OF EDNA ON REGENERATION IN GENETICALLY DYSTROPHIC MOUSE SKELETAL MUSCLE
Section	CONTROLS 'Purina' chow (new fibers/square mm)	Section	TREATED 'Purina' chow+ EDNA {0.2 g/kg} (new fibers/square mm)	F {analysis of variance}
1	5.00	1	12.50
2	5.17	2	15.18
3	5.75	3	13.68
4	11.32	4	15.18
5	6.29	5	16.49
6	5.77	6	16.96
7	5.62	7	13.20
8	7.89	8	11.38
mean-->	6.60+/-1.97 S.D.	mean-->	14.32+/-1.83 S.D.	57.749*
*Normal distribution of (99 %) for 7 d.f. = 7.00.

With each case the belly of the left tibialis anterior was trimmed into a rectanglular block and sectioned tangential to the anterior surface. The data in this table were obtained for randomly selected sections of one control and one experimental case. These sections were examined with a dissecting microscope prior to histological analysis to ensure uniform density and while in place were sized with a stage micrometer. The number of sections in which counts were made constituted approximately 10 % of the total. Tissues were alike with respect to tissue density per unit area, size of non-regenerating muscle fibers and the extent of degeneration.

**Table 4**. INFLUENCE OF EDNA ON REGENERATIVE ACTIVITY AS INDICATED BY THE NUMBERS OF NUCLEI IN MYOTUBES
specimens:	2-3	4-5	6-10	11-15	16-20	21-37
specimens:	-- Nuclei in Myotubes -- {grouped}
Controls	2	45	45	7	1	0
EDNA-treated	13	14	44	16	8	6
Using the specimens described in Table 2 (see legend), ten sections were randomly selected; nuclei were counted in each myotube of each section. Eighty-two myotubes were found in the control sections and 149 in the EDNA-treated specimens. Values were equated to 100 myotubes for simplicity.

**Table 5**. INFLUENCE OF EDNA ON CLIMBING ABILITY OF MICE WITH MUSCULAR DYSTROPHY
Animal Type	N	Diet	Attempts no.	Climbs no.	Climbs %
Normal*	5	'Purina'	10	10	100
Normal*	5	'Purina' + EDNA	10	10	100
DYSTROPHIC**	4	'Purina'	8	2	25
DYSTROPHIC**	5	'Purina' + EDNA	10	9	90
Re 129/Dydy (heterozygous for the trait but without dystrophy) *Re 129/dydy (homozygous for trait) (see ref. 5) Each animal was tested twice. Learning quickly became a variable and a barrier to further rigorous analysis of testing: Treated animals continued to climb sussessfully for the remainder of their lives (which, statistically, were not prolonged by treatment). The untreated animals quickly learned, however, that falling from the test rig bore no untoward consequences (they fell back into the experimenters awaiting hands) and soon made no attempts to sustain themselves (thus falsely exaggerating the successes of the treated animals). The data recorded above represent the first two attempts made by the animals to accomplish the climbing task. The two successful climbs among untreated dystrophic animals were achieved without the use of hind limbs. In contrast, the treated animals were able to flex and extend their hind limbs and always used them in each attempt, both recorded above and in future spot checks while they lived.

**Table 6**. INFLUENCE OF EDNA ON PULLING CAPACITY** OF MYOPATHIC DOGS
Dog	I Weeks 6-13	II Weeks 16-29	III Weeks 30-34	IV Weeks 38-42
Duke{control}	19.7+/-0.54	19.5+/-0.52	20.0+/-0.17	19.3+/-0.85
Pete{control}	18.7+/-0.34	18.9+/-0.46	19.1+/-0.46	18.4+/-0.44
Joe	24.6+/-0.75	23.8+/-3.13	24.8+/-0.80	20.8+/-0.49*
Spike	23.1+/-0.71	22.6+/-1.00	24.0+/-0.48	23.4+/-0.25
Pat	26.4+/-1.37	20.5+/-0.54*	24.0+/-0.98	25.7+/-1.40
Mike	22.6+/-1.30	23.9+/-1.51*	24.8+/-1.39	24.3+/-0.63
Dan	28.1+/-1.26	23.9+/-1.51*	30.3+/-2.04	23.2+/-0.02*
Andy	28.1+/-1.41	27.5+/-3.24	28.3+/-0.51	20.8+/-0.42*
*EDNA treatment in progress for the dog during the indicated period. as measured by time, in seconds** (shown as means for period +/- standard deviation) through a 48 m course pulling a load equal to body weight. Duke and Pete, controls, were on a normal diet throughout the investigation; all others were on the vitamin E deficient ration with deionized drinking water (see text). Dogs were isolated during their entire time in the laboratory. Roman numerals approximate the periods in Table 7 (below).

**Table 7**. INFLUENCE OF EDNA ON STRENGTH OF MYOPATHIC DOGS AS A FUNCTION OF PERCENT CHANGE IN PULLING TIME FOR DIFFERENT PERIODS IN THE INVESTIGATION
Dog	change I vs II {6-15 wks vs 16-24 wks} t (P, 0.01 = 3.06)		change II vs III {16-29 wks vs 30-34 wks} t (P, 0.01 = 5.84)		change III vs IV {30-34 wks vs 38-42 wks} t (P, 0.01 = 5.84)
Dog	% change	t	% change	t	% change	t
Duke	-1	1.39	+1	4.00	-3	1.85
Pete	+1	1.64	+2	1.95	-1	1.53
Joe	+3	0.96	+4	2.80	-16(a)	18.53*
Spike	-2	1.87	+6(b)	6.54*	-2	5.41
Pat	-22(a)	40.97*	+17(b)	8.01*	+7	2.72
Mike	-7(a)	7.00*	+19(b)	6.45	-2	1.78
Dan	-14(a)	10.42*	+26(b)	7.03	-23(a)	15.60*
Andy	-2	0.69	+2	3.52	-26(a)	40.11*
Dogs on EDNA	Pat, Mike, Dan		none		Joe, Dan, Andy
*P< 0.01 (a) Significant increase in strength as indicated by decreased pulling time. (b) Significant decrease in strength as indicated by increased pulling time. Duke and Pete had normal diets throughout, as indicated in Table 6.

Text Footnotes
*Pietsch's present address: Indiana University, Bloomington, Indiana, USA
**EDNA seems to optimize growth without inducing neoplasia or giantism, and its effects are most dramatic when growth conditions are defecient (see ref. 4).

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Web Contact: pietsch@indiana.edu