there is a diversity of chloride channels in mammalian tissues , including ( a ) clc chloride channels , which are often voltage gated , ( b ) gabaa and glycine receptors , which are ligand gated , and ( c ) the cystic fibrosis transmembrane conductance regulator .
the clc chloride channel family in turn consists of many subtypes , including clc-1 through clc-7 plus clc - ka and clc - kb .
these channels differ in cellular location , with clc-1 , clc-2 , clc - ka , and clc - kb residing on the plasma membrane , and the others on intracellular membranes .
genetic defects in these channels cause a number of human diseases , including myotonia congenita , bartter syndrome iii , dent s disease , osteopetrosis , and possibly seizures ( jentsch et al . , 2005 ) .
clc-1 is found predominantly in skeletal muscle ( steinmeyer et al . , 1991a ) .
myotonia congenita in humans results from over 80 different mutations in the clcn1 gene on chromosome 7 .
two clinical forms are recognized , autosomal dominant thomsen s and autosomal recessive becker s myotonia congenita ( koch et al . , 1992 ; ptacek et al . , 1993 ;
mutations of this gene with impaired clc-1 chloride channel function are also found in goats and mice ( heller et al . , 1982 ; watkins and watts , 1984 ; mehrke et al . , 1988 ;
, 1991b ; gurnett et al . , 1995 ; beck et al . , 1996 ; jentsch et al . , 2005 )
reduced chloride channel conductance leads to membrane hyperexcitability , which in turn leads to repetitive electrical discharges following muscle activation .
the net result is a slowing of muscle relaxation as a result of the continued electrical activity ( entrikin et al . , 1987 ;
the myotonia diminishes with repeated muscle contractions , but returns quickly following a period of inactivity ( entrikin et al . , 1987 ;
goats with myotonia develop intermittent muscle stiffening causing them to fall over especially after being startled , whereas mice demonstrate a prolonged stiff leg extensor posture after shaking the cage or dropping the animals from a small height .
some but not all studies have also reported weakness in the intact animal as well as impaired muscle contractile performance when studied experimentally ( heller et al . , 1982 ; watkins and watts , 1984 ;
, 2007a , b ) . the extent to which clc-1 chloride channel deficiency affects exercise performance is controversial .
descriptions of human disease typically mention impairment of exercise performance , in particular at or near the onset of exercise , although there are also reports of subjects who engage in vigorous exercise ( entrikin et al . , 1987 ; burnham , 1997
; weinberg et al . , 1999 ; stimson , 2001 ; colding - jorgensen , 2005 )  however controlled quantitative data are scant .
the original report of myotonic mice notes that the animals walk almost normally , albeit more slowly and slightly stiffly , and are able to swim albeit with some degree of hindleg uncoordination ; only when placed in an ice - water bath does the impairment in swimming become prominent , although quantitative data are lacking ( heller et al . , 1982 ) .
in contrast , there is one quantitative study which documented a very large ( 6065% ) reduction in spontaneous locomotor activity of myotonic mice while in a cage ( heimann et al . , 1988 ) . the latter data , however , most likely reflects predominantly low distance walking rather than moderate to high distance walking and running . based on the above information ,
it is unclear whether clc-1 deficiency has mild or severe adverse effects on exercise activity .
the purpose of the present study was to test the hypothesis that skeletal muscle clc-1 chloride channel deficiency leads to severe impairments in spontaneous exercise , which was tested in myotonic mice by assessing voluntary wheel running .
voluntary wheel running was chosen as the model for a moderate to high amount of exercise because normal rodents typically spontaneously walk and run 410  km / day when provided access to running wheels ( dupont - versteegden et al . , 1994 ; carter et al .
, 1995 ; hayes and williams , 1996 ; van lunteren and moyer , 2003 ; van lunteren et al . , 2004 ) .
furthermore , as noted previously , descriptions of the manifestations of the myotonia differ among humans , goats , and mice .
in contrast , voluntary walking and running is a form of exercise that is common to both mice and humans , so that the findings in myotonic mice will be directly applicable to humans with myotonia congenita .
all studies were approved by the institutional animal care and use committee at the cleveland department of veterans affairs medical center ( cleveland , oh , usa ) , and performed in accordance with nih animal care guidelines .
male homozygous ( / ) myotonic mice ( swr / j - clcn1/j ; / ; n  =  3 ) and phenotypically normal control animals (  or + /+ ; n  =  4 ) were obtained from jackson laboratories ( bar harbor , me , usa ) . throughout their lifetime heterozygous (  ) mice are behaviorally indistinguishable from homozygous ( + /+ ) normal controls ; furthermore , the electrical activity of heterozygotes can not be distinguished from homozygous normal controls ( heller et al . , 1982 ) .
thus many studies of myotonic mice group heterozygous (  ) and homozygous ( + /+ ) mice together as controls ( heller et al . , 1982 ; fuchtbauer et al . , 1988 ; mehrke et al . , 1988 ; goblet and whalen , 1995 ; kramer et al . , 1998 ; van lunteren et al .
, 2007a , b ) , similar to the present study . at the time of entry into the study
assessment of spontaneous running was done using standard size polycarbonate living chambers which contained running wheels ( van lunteren and moyer , 2003 ; van lunteren et al . , 2004 ) .
the running wheels were equipped with a magnetic counter , the output of which was sent to a pc - type computer ( lafayette instruments , lafayette , in , usa ) .
this allowed quantification of the number of revolutions , and multiplying this by the circumference of the running wheels provided measurements of distance .
the animals spent 8  weeks in running wheel - equipped housing , and were given ample access to food and water 24  h / day .
recordings of the number of rotations of each running wheel were performed 24  h / day for the entire 8  week period . the total distance each animal ran per day
, 2002 ; van lunteren et al . , 2004 ) of identifying active running times was adopted ; specifically any 10  min interval during which the animals ran enough to produce at least one rotation of the wheel was defined as an active running period .
average consecutive running times were calculated from the number of 10  min periods in a row during which the animals ran .
thus the consecutive running time indicates the total duration of time during which the animals ran enough to produce at least the pre - defined minimum number of wheel rotations during each 10  min interval , and does not imply that the animals ran without stopping the entire time ( hara et al . , 2002 ; van lunteren et al . , 2004 ) . in previous studies of normal rats we found that when animals are provided access to a running wheel , they gradually increase their running distance over the course of 35  weeks and then remained relatively stable thereafter ( van lunteren and moyer , 2003 ; van lunteren et al . , 2004 ) .
statistical analysis was performed with the unpaired t - test for comparisons of two data sets .
analysis of variance , followed by the student - newman  keuls test in the event of a significant analysis of variance result , was used for statistical testing of multiple data sets . a p value of < 0.05 ( two - tailed ) was chosen to indicate significance .
the myotonic mice used for the running studies weighed considerably less than the wild type mice ( 12.7    0.3 vs 25.5    0.3  g ) , as has also been noted in previous studies of this mouse model ( heller et al . , 1982 ; heimann et al . , 1988 ;
delayed muscle relaxation during isometric contractions was verified in previous muscle contractile studies of clc-1 chloride channel - deficient myotonic animals ( heller et al . , 1982 ; heimann et al . , 1988 ; mehrke et al . , 1988 ; reininghaus et al . , 1988 ; van lunteren et al . , 2004 , 2007b ) .
examples of the temporal pattern of running over the course of each day during the eighth week are depicted in figure 1 .
both normal and myotonic mice did the vast majority of their wheel running between 8 pm and 8 am , as expected for nocturnal animals .
normal mice almost always ran for multiple consecutive 10-min periods , typically 1520 consecutive periods at the onset of the night , and then fewer consecutive periods as the night went on .
in contrast , myotonic mice only ran only a few times per night , and for short periods of time per episode ( typically no more than three consecutive 10-min periods ) .
example of running behavior of a normal and myotonic mouse during all of week 8 .
the distance run is quantified for each 10  min interval as a function of time during the day .
each day depicted is from 8 am one morning until 8 am the following morning .
data were obtained and averaged for all 7  days of the eighth week of running ( figure 2 ) .
in addition , all normal animals averaged more than 1300  m / day ( with most running in excess of 5000  m / day ) , whereas all myotonic mice averaged less than 700  m / day .
the mean consecutive running time was significantly shorter for myotonic than normal mice ( figure 2b ) .
changing the threshold for defining the consecutive running time from at least one revolution to a higher value did not change the finding of shorter consecutive running times in myotonic compared with control animals , as depicted in figure 2c for a threshold of 3  m/10  min interval .
similarly , the average consecutive running distance ( defined as the total distance run during each consecutive running period ) was lower for myotonic than control mice ( figure 2d ) .
data are depicted for individual animals ( small circles ) as well as mean values for each group of animals ( se ) .
asterisks indicate statistical significance between normal and myotonic mice ( * p  =  0.048 , * * p  =  0.025 , * * * p  <  0.001 ) .
( a ) total running distances per day ; ( b ) average consecutive running times based on any running at all during the 10  min period ; ( c ) average consecutive running times quantified based on the animals having to have run at least 3  m during the 10  min period to be considered as having run ; ( d ) average consecutive running distances based on any running during the 10-min period . the time course of changes in running distance of normal and myotonic mice over the 8-week period is depicted in figure 3a . during the first few days of access to the running wheels , the normal mice gradually increased the amount of running . at 2  weeks
the animals averaged > 4  km / day , and this distance was maintained thereafter ( albeit with a slight downward trend ) .
in contrast , the myotonic mice did not increase the running distance from the first few days to later time periods .
running distances were significantly greater for normal than myotonic mice at 2 , 4 , and 8  weeks .
changes in average daily running distances ( a ) and average consecutive running times ( b ) for normal and myotonic mice as a function of time after being provided access to the running wheels .
the 2 , 4 , and 8-week data were quantified during the last 2  days of the week .
the above temporal patterns seen with total running distance were also noted for consecutive running time ( figure 3b ) .
myotonic mice had significantly lower consecutive running times than normal mice at 2 , 4 , and 8  weeks , but not at 23  days .
examples of the temporal pattern of running over the course of each day during the eighth week are depicted in figure 1 .
both normal and myotonic mice did the vast majority of their wheel running between 8 pm and 8 am , as expected for nocturnal animals .
normal mice almost always ran for multiple consecutive 10-min periods , typically 1520 consecutive periods at the onset of the night , and then fewer consecutive periods as the night went on .
in contrast , myotonic mice only ran only a few times per night , and for short periods of time per episode ( typically no more than three consecutive 10-min periods ) .
example of running behavior of a normal and myotonic mouse during all of week 8 .
the distance run is quantified for each 10  min interval as a function of time during the day .
each day depicted is from 8 am one morning until 8 am the following morning .
data were obtained and averaged for all 7  days of the eighth week of running ( figure 2 ) .
in addition , all normal animals averaged more than 1300  m / day ( with most running in excess of 5000  m / day ) , whereas all myotonic mice averaged less than 700  m / day .
the mean consecutive running time was significantly shorter for myotonic than normal mice ( figure 2b ) .
changing the threshold for defining the consecutive running time from at least one revolution to a higher value did not change the finding of shorter consecutive running times in myotonic compared with control animals , as depicted in figure 2c for a threshold of 3  m/10  min interval .
similarly , the average consecutive running distance ( defined as the total distance run during each consecutive running period ) was lower for myotonic than control mice ( figure 2d ) .
data are depicted for individual animals ( small circles ) as well as mean values for each group of animals ( se ) .
asterisks indicate statistical significance between normal and myotonic mice ( * p  =  0.048 , * * p  =  0.025 , * * * p  <  0.001 ) .
( a ) total running distances per day ; ( b ) average consecutive running times based on any running at all during the 10  min period ; ( c ) average consecutive running times quantified based on the animals having to have run at least 3  m during the 10  min period to be considered as having run ; ( d ) average consecutive running distances based on any running during the 10-min period .
the time course of changes in running distance of normal and myotonic mice over the 8-week period is depicted in figure 3a . during the first few days of access to the running wheels ,
the animals averaged > 4  km / day , and this distance was maintained thereafter ( albeit with a slight downward trend ) .
in contrast , the myotonic mice did not increase the running distance from the first few days to later time periods .
running distances were significantly greater for normal than myotonic mice at 2 , 4 , and 8  weeks .
changes in average daily running distances ( a ) and average consecutive running times ( b ) for normal and myotonic mice as a function of time after being provided access to the running wheels .
the 2 , 4 , and 8-week data were quantified during the last 2  days of the week .
the above temporal patterns seen with total running distance were also noted for consecutive running time ( figure 3b ) .
myotonic mice had significantly lower consecutive running times than normal mice at 2 , 4 , and 8  weeks , but not at 23  days .
not only was the total distance run per day reduced substantially , but the animals ran for shorter consecutive durations , and for shorter distances each time they ran . furthermore , normal mice gradually increased the running distance and consecutive running times when first provided access to the running wheels , whereas myotonic mice did not .
one issue about the design of the present study is the use of relatively small sample sizes of three myotonic mice and four normal mice .
large differences in running distances and times were noted between myotonic and normal animals which greatly exceeded variability in values within each group , resulting in statistically significant findings .
calculations were performed on the 8-week data to determine whether the study was adequately powered to have at least an 80% chance of detecting significant differences at a p  <  0.05 level .
the following power values were found : 96% for average running distance , 100% for average consecutive running time with no threshold , 89% for average consecutive running time with a 3-m threshold , and 86% for average distance per running period .
thus power analysis indicates that the sample sizes were indeed adequate to statistically detect the large differences in running parameters between myotonic and control mice .
many descriptions of exercise capacity in humans with myotonia congenita are qualitative rather than quantitative ( ptacek et al . , 1993 ; burnham , 1997 ; weinberg et al . , 1999 ; stimson , 2001 ; colding - jorgensen , 2005 ) or compare myotonic subjects before and after an intervention rather than myotonic vs normal subjects ( birnberger et al .
, 1975 ; hammaren et al . , 2005 ) . impaired ability to perform athletic activities is described frequently , in particular for strenuous activities . on the other hand , various degrees of impairment can be overcome by engaging in a suitable antecedent warm - up period , and furthermore the time course over which myotonia dissipates is considerably faster than the time course over which fatigue develops during high intensity repetitive contractions ( van lunteren et al . , 2011 ) . as a result ,
some subjects with myotonia congenita are able to engage successfully ( albeit not always fully normally ) in strenuous athletic activities ( burnham , 1997 ; weinberg et al . , 1999 ;
1982 ) noted that the animals walk almost normally , but on closer inspection their gait is slightly  stiff  and walking is slower than unaffected littermates .
( 1988 ) video - recorded mice while on a plane surface , and found that the relative reduction in spontaneous activity of myotonic mice ( 6065% ) was greater than the magnitude of reduced activity ( 5055% ) seen in dystrophin - deficient mdx dystrophic mice ( an animal model of human duchenne muscular dystrophy ) .
although the running activity of myotonic mice has not been reported previously , that of dystrophin - deficient mdx dystrophic mice has been quantified .
( 1994 ) found that control mice ran 7  km / day whereas mdx dystrophic mice ran 5  km / day .
( 1995 ) found no significant running deficit in the young ( 4  week old ) dystrophic mice but a 5070% reduced distance run in adult ( 6  month old ) dystrophic mice .
hayes and williams ( 1996 ) found average running distances of 30  km / week in mdx dystrophic mice and 45  km / week in control mice .
the present study found that myotonic mice had deficits in running distance of 8590% , thus exceeding the above reported reductions seen in mdx mice .
hayes and williams ( 1996 ) also reported on the running behavior of 2-laminin - deficient dy / dy dystrophic mice ( an animal model of human classic congenital muscular dystrophy ) .
the dy / dy mice have greater generalized weakness than mdx mice ( carter et al . , 1995 ; jentsch et al . ,
2005 ) , and this was reflected in very large impairments of running distance ( 8095% ) .
therefore the running activity deficit of myotonic mice is comparable to that of dy / dy dystrophic mice .
( 2002 ) found shortened continuous running times in the mdx dystrophic compared with control animals ( 2030 vs 5080  min , depending on age ) .
a running endurance abnormality also occurs in streptozotocin - induced diabetes , with average values at 8  weeks of 16 and 40  min in diabetic and normal rats , respectively ( van lunteren et al . ,
the deficit in the average consecutive running time of the myotonic animals was in the 6070% range , and thus comparable in magnitude to the deficits seen in both mdx dystrophic mice and diabetic rats .
several mechanisms may contribute to the impaired running activity ( present study ) and activity levels ( heimann et al .
first , the myotonic mice were smaller than the normal mice despite comparable ages , in agreement with previous reports of this mouse model ( heller et al . , 1982 ;
reduced weight due to fat loss should improve running , whereas reduced weight due to sarcopenia should reduce running .
second , it is possible that the animals did not want to run instead of that they could not run , similar to what has been reported with hormonal interventions .
steinmeyer et al . ( 1991a ) examined the tissue distribution of clc-1 by northern blot analysis .
there was a prominent band in skeletal muscle , but faint bands in kidney , liver , heart , and a smooth muscle cell line .
thus clc-1 is found predominantly , but probably not exclusively , in skeletal muscle making it relatively unlikely that it could affect a mouse s motivation to run .
third , the myotonic mice have substantial electrical and mechanical myotonia as a direct effect of the clc-1 chloride channel loss , as reported previously ( heller et al . , 1982 ; heimann et al . , 1988 ;
mehrke et al . , 1988 ; reininghaus et al . , 1988 ; van lunteren et al . , 2004 , 2007b ) , and this certainly will impair muscle performance during intermittent contractions .
fourth , muscle from humans and animals with myotonia congenita has structural , biochemical , and contractile alterations which go beyond the direct effects of the markedly reduced or absent clc-1 channels .
this includes alterations in fiber type composition and myosin isoform distribution ( in particular loss of type iib fibers and myosin ) , down regulation of parvalbumin , and abnormal expression of myogenic regulatory factors ( myod and myogenin ; stuhlfauth et al . , 1984 ; reininghaus et al . , 1988 ; agbulut et al . ,
in addition , variable degrees of weakness have been noted in the intact animal ( heller et al . , 1982 ;
watkins and watts , 1984 ) , and studies of isolated muscles have noted impaired performance during the contraction phase of the contraction 
the extent of impairment is considerable , for example isometric force is reduced by 3565% whereas isotonic power and work impairments may exceed 50% ( van lunteren et al .
fifth , there may have been a training response during the course of the 8-weeks of running in the normal mice which exceeded that of the myotonic mice .
however , both groups of mice were housed in conventional cages prior to the onset of the 8-week running period , and therefore the early difference between groups can not be attributed to training responses . finally , it is unlikely that pain contributed to differences between groups , as there was no evidence for the myotonic mice having pain while running , and we are not aware of pain being a significant issue in this disease .
it is interesting to consider whether exercise training might revert the effects of the clc-1 chloride channel deficiency .
if there was an improvement in muscle function with repetitive running one would expect that the myotonic animals should increase the amount of running during the 8-weeks of study ; rather there was actually a small decrease over time .
the present study used voluntary rather than forced exercise , and thus assessed the extent to which exercise gets performed on a regular basis rather than maximum exercise capacity . forced
exercise regimens in rodents typically involve application of noxious or painful stimuli ( e.g. , electrical shocks ) to ensure that the animals effort is maximal
. the resultant stress may produce substantial physiological responses that differ from those of exercise itself .
humans participate in highly structured exercise regimens during athletic or military training , but frequently exercise performance is encouraged verbally rather than enforced by noxious or painful stimuli .
therefore , data from a myotonic rodent model of forced exercise would be less applicable to myotonic humans than a rodent model of voluntary exercise . on the other hand forced treadmill running
might have allowed us to test whether a warm - up period improves exercise performance .
in conclusion , clc-1 chloride channel - deficient myotonic mice have deficits in spontaneous exercise performance .
the magnitude of the deficit is quite large , and in many respects as great as that seen in animal models of muscular dystrophy .
one limitation of the present study with respect to extrapolating these findings to humans with myotonia congenita is that myotonia dissipates with repeated contractions ( the  warm - up phenomenon  ) , and thus it may be possible for humans with clc-1 deficient myotonia to overcome at least part of the exercise deficit by a well - designed warm - up regimen prior to initiating exercise .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 background : genetic deficiency of the muscle clc-1 chloride channel leads to myotonia , which is manifested most prominently by slowing of muscle relaxation . 
 humans experience this as muscle stiffness upon initiation of contraction , although this can be overcome with repeated efforts ( the  warm - up  
 phenomenon ) . the extent to which clc-1 deficiency impairs exercise activity is controversial . 
 we hypothesized that skeletal muscle clc-1 chloride channel deficiency leads to severe reductions in spontaneous exercise . 
 methodology / principal findings : to examine this quantitatively , myotonic clc-1 deficient mice were provided access to running wheels , and their spontaneous running activity was quantified subsequently . 
 differences between myotonic and normal mice in running were not present soon after introduction to the running wheels , but were fully established during week 2 . during the eighth week 
 , myotonic mice were running significantly less than normal mice ( 322    177 vs 5058    1253  m / day , p  =  0.025 ) . 
 furthermore , there were considerable reductions in consecutive running times ( 18.8    1.5 vs 59.0    3.7  min , p  <  0.001 ) and in the distance per consecutive running period ( 58    38 vs 601    174  m , p  =  0.048 ) in myotonic compared with normal animals . 
 conclusion / significance : these findings indicate that clc-1 chloride deficient myotonia in mice markedly impairs spontaneous exercise activity , with reductions in both total distance and consecutive running times . 
Introduction
Materials and Methods
Results
Running activity at 8 weeks
Time course over which changes in running occurred
Discussion
Conflict of Interest Statement