The inability of muscles to relax normally after voluntary contractions is characteristic of the disease myotonia. The results of this study showed that mechanical myotonia decreases rapidly during fatigue-inducing stimulation. Furthermore, myotonia was found to decrease faster than force in both the diaphragm and EDL muscles. The normalization of relaxation times was slightly faster at a higher frequency (50 Hz) than a lower frequency (20 Hz) in the diaphragm muscle; however this may have been due to the effects of myotonia being less during 50 Hz than 20 Hz stimulation. Furthermore, the resolution of myotonia was also slightly faster in the EDL muscle than the diaphragm muscle, although again possibly because of differences in the magnitude of the myotonia. Both the diaphragm and EDL muscles had much larger degrees of myotonia than the soleus in response to 9-AC. In addition, force production increased at the lower stimulation frequency (20 Hz) in the diaphragm and EDL, and increased slightly in the soleus at this frequency, but did not increase at the higher frequency (50 Hz) in any of the three muscles with 9-AC. Contraction time increased only in the diaphragm during 20 Hz stimulation.
Myotonia, whether genetically- or drug-induced, decreases during repeated contractions, this resolution being termed the "warm up" phenomenon. This has been described in clinical reports of humans with myotonia congenita and is also apparent when observing the behavior of genetically myotonic mice [2
]. Physiological manifestations when tested experimentally include improved rate of muscle relaxation and diminution of the duration and intensity of persistent electromyographic activity following repetitive muscle activation.
One of the early experimental studies of the warm up phenomenon was that performed by Senges and Rudel [16
] in a model of myotonia induced by 2,4-dichlorphenoxyacetate. They examined the effects of a conditioning tetanus preceding a test contraction by 0.5, 1, 2 and 4 seconds. The shortest time interval virtually abolished myotonia, with the effect diminishing markedly as the time interval increased.
The warm up phenomenon was described by Heller et al.[7
] in the original report of genetically myotonic mice. This was demonstrated based on electromyographic recordings from affected muscle in response to direct peripheral nerve stimulation rather than on measurements of muscle force. Heller and colleagues [7
] described the myotonia as declining with repeated trials at 10 second intervals, but recovering when the muscle has rested for one minute.
The warm-up phenomenon was also studied by Heimann et al [27
]. EDL muscles from myotonic ADR mutants and ADR-MDX double mutants were shown to have similar degrees of myotonia as quantified by the myotonia index [28
] (myotonia index of 0.49 vs. 0.57, respectively). Furthermore, they had similar degrees to which the myotonia improved when contractions were preceded by a series of single twitches and incomplete tetanic stimulations. However, the study also found that myotonia symptoms were more pronounced in ADR than ADR-MDX muscle, but ADR-MDX mice had higher levels of weight reduction and premature death. In contrast, for the soleus the ADR mutant had a significantly higher myotonia index than the ADR-MDX mutant (myotonia index of 0.90 vs. 0.61). Data for the warm up phenomenon were not depicted for the soleus.
Van Beekvelt et al. [21
] studied three humans with myotonia and defined the warming-up phenomenon as the force recovery phase after initial paresis during a sustained voluntary contraction. This study hypothesized that warming-up was due to the enhanced activation of Na+-K+-ATPase during exercise, and used ouabain (a Na+-K+-ATPase inhibitor) to test this. However, it was found that ouabain infusion did not prevent recovery from transient paresis. Therefore, it was concluded that the warm-up phenomenon was not due to Na+-K+-ATPase.
Taken together, the above studies provide a comprehensive picture of the manner in which myotonia improves following brief contractions, but these studies had not examined changes in myotonia during the course of repetitive fatigue-inducing contractions. The latter issue has been examined to a limited extent in a previous study from our lab in which we examined isotonic contractions of diaphragm muscle from genetically myotonic mice [19
]. Diaphragm from these mice had lesser degrees of myotonia than seen in the diaphragm of the present study. Total relaxation time during a singe train contraction ranged from ~0.2 to 0.6 seconds in contrast to values exceeding 2 seconds with 9-AC in the present study. In the genetically myotonic mice, repetitive train stimulation at a frequency of 50 Hz resulted in a rapid reduction in relaxation time over the course of 30 seconds, with minimal changes thereafter during the subsequent 30 seconds.
The present study expands on our previous findings [19
] in several manners. We found that the reduction of myotonia during fatigue-inducing contractions occurs during isometric as well as isotonic contractions, and in the drug-induced myotonia model as well as genetic myotonia. Furthermore, the present study indicates that the resolution of myotonia varies as a function of the frequency of muscle stimulation. In addition the extent of drug-induced myotonia differed among the three muscles studied, with the slow fiber type predominance of the soleus appearing to have had a protective effect. Finally, the present data indicate that the time course of the warm-up phenomenon is much faster than that of force loss during fatigue-inducing contractions.
One of the limitations of the present study is that the studies were done in vitro, under conditions in which there is no blood flow and thus no delivery of nutrients and/or impaired removal of potentially adverse metabolites. Thus the development of fatigue may have occurred faster in the drug-induced myotonia model than would occur in vivo in humans or animals with genetic CLC-1 chloride channel deficient myotonia. A second limitation is that the genetic muscle diseases that cause myotonia are a heterogeneous group which include not only two variants of CLC-1 deficient myotonia but also myotonic dystrophy. Several of these disorders have features in addition to the myotonia, which for some includes muscle weakness. Among these additional features, muscle weakness in particular may impact the rate at which fatigue develops, thereby changing the temporal relationship between the improvement of the myotonia and the reduction of force in response to repetitive vigorous contractions.