Biochemical Research Laboratory, The Dow Chemical Company, Midland, Michigan
Ethylenedinitramine (EDNA), a plant growth stimulator (1) and high impact explosive, ameliorates the symptoms of dietary and hereditary muscular dystrophy in laboratory animals (2). These beneficial effects have been correlated with enhanced muscle regeneration. In mechanically injured muscle, EDNA increases the regenerative yield most dramatically when its administration is time to coincide with certain critical periods (3) before the new fibers appear. We speculated that sub-anesthetic concentrations of ethylene, too, might increase muscle regeneration.ABSTRACT
In mice, ethylene inhalation enhanced skeletal muscle regeneration in heredity muscular dystrophy and following mechanical injury.
Wounded muscle was studied using 3-month C57BL/6J males with birthdays +/- 3 days of each other. Aseptic incisions, 1.5 x 2 mm, were inflicted in the belly of the tibialis anterior, 3 mm from the origin. Exposures to air or ethylene were carried out in Saran enclosures as depicted in Fig. 1 (see legend for details) and summarized in Fig. 2. Groups of animals received:
Alternating 12-hr periods of light and darkness are maintained in our colony as a standard procedure. New animals are acclimatized to this rhythm for a minimum of 2 weeks. In the present studies all animals had been maintained on the same light-dark regimen for 1 month prior to experimentation. The same schedule was maintained throughout the investigation. The importance of controlling light-dark rhythm in muscle regeneration is discussed elsewhere (see pp. 3-4, Ref. 3).
The EDNA molecule interacts in vivo with chromatin; RNA and protein moieties appear to contribute to the integrity of the reaction product (9). Ethylene, in turn, reacts with RNA (10). Both molecules bind metals (8, 11). Given a ligand, metal-substituted ethylene can polymerize, and the same is probably true of EDNA. Ethylene and EDNA might, as a result of metal binding, form ligand-dependent oligomers that:
At anesthetic concentrations ethylene can induce leukopenia (12); but in the low range, as employed in our experiments, no leukocytic changes occur after as long as 90 continuous days of exposure (13). Inhibition of cholinesterase activity was reported in the latter work; this observation might parallel the well-known ripening effects of ethylene on plants (14).
We bring up these points because some of our experiments indicated that ethylene had negative as sell as positive effects on muscle regeneration. This was not immediately obvious and only became evident upon careful comparison of values for continuous and discontinuous schedules:
While 5.7 % ethylene inhalation stimulated an increase in myotube formation in each of the schedules, the comparative results do not reflect simple addition. Continuous exposure was appreciably less efficient than the discontinuous nighttime regimen. Simple addition would require that the values from continuous experiments be greater than or, at the minimum, equal to the nighttime values. The number of myotubes that developed in response to continuous inhalation, therefore, must contain a negative component, hidden in the overall positive character of the final outcome. It is convenient to regard the results of each schedule as representing plus and minus terms summed algebraically over a range from zero to some positive value. Then, reasoning from enhancement in each schedule, the pluses outweighed the minuses and neither discontinuous schedule contained all the elements represented in continuous exposure; nighttime was deficient in minus components and the daytime regimen did not reflect a full complement of pluses. The logic of this is easily accommodated on temporal grounds, and it follows quite simply that plus and minus factors occur at different periods in early regeneration. Early regeneration -- the interval before myotubes appear -- is by no means a homogeneous phase of the process. Different events correspond to the nighttime and daytime inhalation schedules. Inhalation of ethylene during the nighttime schedule occurred during waves of DNA synthesis (3). In the daytime schedule ethylene "missed" the waves of replication but "hit" mitosis per se (7) as well as an interval (around 72 hr) of extreme sensitivity to actinomycin D (4) [presumably a period of intense messenger RNA synthesis].
The point made in the last paragraph is not that ethylene exerts its net effect on regeneration by enhancing DNA synthesis and suppressing transcription or mitosis, but that these well-characterized initial phases of the regenerative process are, at the minimum, concomitants of whatever it is that ethylene affects pharmacodynamically. Events such as replication, mitosis, etc. can serve as useful guideposts in designing future investigations. Given, for example, a comprehensive knowledge of synchrony in dystrophic muscle, a schedule might be devised to enhance regeneration several orders beyond those we observed.
Fig. 1. Schematic illustration of exposures: air was driven into the system by pumps and ethylene, USP grade, from a cylinder; cages were enclosed in Saran bags; gas flow was regulated in each line by a regulator valve, not shown in the illustration. Each flow meter had been calibrated previously. The concentration of ethylene was determined both from samples taken at the sample port and from the various void outlets. It was necessary to employ a micropump to deliver the 0.1% concentration of ethylene illustrated in the upper right. Tubing was of Tygon, joints of polypropylene, and sealing was accomplished with vinyl tape; electrical connections were sealed to minimize fire hazards. The concentrations of ethylene were far below the anesthetic range.
Fig. 2 (wounded muscle only)
| parameter | Control | Ethylene |
|---|---|---|
| Sample size | 11 | 6 |
| Sample mean (+/- SD) | 0.114 (0.05)* | 0.408 (0.06)* |
| Variance | 0.002 | 0.003 |
| SE of mean | 0.015 | 0.026 |
| Confidence limits** (99.99 level) | 0.08-0.20 | 0.31-0.51 |
*regenerating myotubes/nonregenerating muscle fibers
**for a population asymptotically approaching infinity.
| GAS* | Regeneration 100 x myotubes/cut fibers | Analysis of Variance*** (significance level) versus |
||
|---|---|---|---|---|
| Day | Night | means (+/- SD)** | Air alone | Ethylene at night |
| Air | Air | 103 (16) | -- | 0.01 |
| 0.1 | 0.1 | 101 (06) | insignificant | 0.01 |
| 5.7 | 5.7 | 156 (10) | 0.01 | 0.01 |
| 5.7 | Air | 123 (18) | 0.05 | 0.01 |
| Air | 5.7 | 240 (76) | 0.01 | -- |