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Experimental Physiology 92.4 pp 717-729
DOI: 10.1113/expphysiol.2007.038190
© The Physiological Society 2007
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Isotonic contractile impairment due to genetic CLC-1 chloride channel deficiency in myotonic mouse diaphragm muscle

 Erik van Lunteren1,2, Jennifer Pollarine1 and Michelle Moyer2

1 Pulmonary and Critical Care Medicine, Case Western Reserve University, Cleveland, OH, 44106, USA 2 Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, OH 44106, USA


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The hallmark of genetic CLC-1 chloride channel deficiency in myotonic humans, goats and mice is delayed muscle relaxation resulting from persistent electrical discharges. In addition to the ion channel defect, muscles from myotonic humans and mice also have major changes in fibre type and myosin isoform composition, but the extent to which this affects isometric contractions remains controversial. Many muscles, including the diaphragm, shorten considerably during normal activities, but shortening contractions have never been assessed in myotonic muscle. The present study tested the hypothesis that CLC-1 deficiency leads to an impairment of muscle isotonic contractile performance. This was tested in vitro on diaphragm muscle from SWR/J-Clcn1adr-mto/J myotonic mice. The CLC-1-deficient muscle demonstrated delayed relaxation, as expected. During the contractile phase, there were significant reductions in power and work across a number of stimulation frequencies and loads in CLC-1-deficient compared with normal muscle, the magnitude of which in many instances exceeded 50%. Reductions in shortening and velocity of shortening occurred, and were more pronounced when calculated as a function of absolute than relative load. However, the maximal unloaded shortening velocity calculated from Hill's equation was not altered significantly. The impaired isotonic contractile performance of CLC-1-deficient muscle persisted during fatigue-inducing stimulation. These data indicate that genetic CLC-1 chloride channel deficiency in mice not only produces myotonia but also substantially worsens the isotonic contractile performance of diaphragm muscle.

(Received 24 April 2007; accepted after revision 26 April 2007; first published online 4 May 2007)
Corresponding author E. van Lunteren: Pulmonary Section, 111 J(W), Louis Stokes Cleveland Department of Veterans Affairs Medical Center, 10701 East Boulevard, Cleveland, OH 44106, USA. Email: exv4{at}cwru.edu


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
A number of human muscle diseases are characterized by myotonia, including Thomsen's and Becker's myotonia congenita, myotonic dystrophy, paramyotonia congenita and some types of hyperkalaemic periodic paralysis (Ptacek et al. 1993). Prominent muscle wasting and weakness sets myotonic dystrophy apart from the other disorders. Perturbations of Na+ channels underlie many of the hyperkalaemic periodic paralyses, whereas disturbances of Cl– channels underlie myotonia congenita and myotonic dystrophy (Koch et al. 1992; Ptacek et al. 1993; Kleopa & Barchi, 2002; Pusch, 2002; Jentsch et al. 2005; Puljak & Kilic, 2006). Thomsen's and Becker's myotonia congenita are both diseases of CLC-1 (membrane-spanning skeletal muscle channels which help to regulate muscle contractions) chloride channels, with the former having autosomal dominant and the latter autosomal recessive inheritance. The forms of myotonia in goats and mice are also inherited CLC-1 chloride channel disorders (Heller et al. 1982; Watkins & Watts, 1984; Mehrke et al. 1988; Reininghaus et al. 1988; Steinmeyer et al. 1991; Gurnett et al. 1995; Beck et al. 1996; Jentsch et al. 2005).

The hallmark of muscle function in the CLC-1-deficient myotonias is persistent electrical activity following a contraction, resulting in delayed relaxation. Underlying these abnormalities is reduced membrane Cl– conductance, leading to membrane hyperexcitability. Genetically CLC-1 chloride channel-deficient myotonic muscle is characterized by the near or total absence of type IIB fibres, but relatively minor histological changes otherwise (Heller et al. 1982; Reininghaus et al. 1988). From a functional perspective, humans with these disorders experience muscle stiffness which interferes with muscle contraction; this impairment eases with repeated contractions, the so-called ‘warm-up’ phenomenon (Ptacek et al. 1993). Respiratory problems, including ‘breathing difficulties’, dyspnoea, sleep apnoea and daytime alveolar hypoventilation, have been reported in human myotonia congenita (Garcin et al. 1967; Harel et al. 1979; Striano et al. 1983; Estenne et al. 1984). Myotonic goats are described as having impaired ability to jump and climb fences, whereas myotonic mice walk slowly and stiffly but have relatively normal swimming performance (Heller et al. 1982; Watkins & Watts, 1984).

It is not clear to what extent impaired function in humans and animals is due to the myotonia alone versus myotonia plus impaired force generation independent of the myotonia. Based on clinical descriptions of the ADR myotonic mouse, there are conflicting reports that weakness is not a prominent feature (Heller et al. 1982), as well as that these animals have progressive weakness (Watkins & Watts, 1984). Lowering the extracellular chloride concentration affects fatigue resistance of normal muscle (Esau & Sperelakis, 1986; De Luca et al. 1990; Cairns & Dulhunty, 1995; Cairns et al. 2004), as does pharmacological blockade of Cl– channels (De Luca et al. 1990). The well-documented changes in fibre type and myosin isoform composition of myotonic humans and mice (Heene et al. 1986; Reininghaus et al. 1988; Agbulut et al. 2004) would be expected to alter muscle performance not only during relaxation but also during contraction. However, the extent to which myotonic muscle has deficits in isometric contractile performance remains controversial (Entrikin et al. 1987; Reininghaus et al. 1988; van Lunteren et al. 2007).

The above studies examining effects of altered chloride concentrations, pharmacological blockade of chloride channels and genetic CLC-1 chloride channel deficiency were all performed during isometric contractions. The diaphragm shortens considerably during breathing, as do many limb muscles during locomotion, hence contractile performance during shortening contractions has a large impact on the ability to breathe and walk. Isometric and isotonic contractions differ importantly at a cellular level, for example with respect to cross-bridge formation and energetic requirements, so that isotonic contractile performance cannot necessarily be predicted from data obtained under isometric conditions. The hypothesis of the present study is that CLC-1 chloride channel deficiency has an adverse effect on the isotonic performance of diaphragm muscle during the contractile phase of the contraction–relaxation cycle.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The mouse model used for these studies is the myotonic arrested development of righting response (ADR) mouse, in which the CLC-1 chloride channel defect is transmitted in an autosomal recessive manner. Homozygous male ADR myotonic mice (SWR/J-Clcn1adr-mto/J; n = 14) and their phenotypically normal (+/?) controls (n = 15) were purchased from Jackson Laboratories (Bar Harbour, ME, USA), given free access to food and water, and studied at an age of 6 weeks. The age chosen for study is in line with previous investigations of this model (Entrikin et al. 1987; Fuchtbauer et al. 1988; Reininghaus et al. 1988; van Lunteren et al. 2007) and is sufficiently old that the myotonia is already well established (Entrikin et al. 1987; Fuchtbauer et al. 1988; Reininghaus et al. 1988). All procedures were approved by the local institutional animal care and use committee, and followed NIH animal welfare guidelines. Anaesthesia was induced with an intraperitoneal injection of rodent anaesthetic cocktail (initial dose, ketamine 21–30 mg kg–1, xylazine 4.3–6.0 mg kg–1 and acepromazine 0.7–1.0 mg kg–1, with supplemental smaller doses given as needed to produce and maintain a deep level of anaesthesia). Subsequently, the diaphragm was surgically removed and placed in an oxgyenated bath of physiological solution. The connective tissue at the margin of the ribcage and the central tendon of the diaphragm were kept intact at the time of removal. Muscle strips of ~3 mm width were dissected, surgical thread was tied to the rib and central tendon ends, and the diaphragm samples were mounted vertically in a double-jacketed bath. Studies were performed in oxygenated (95% O2–5% CO2) physiological solution, which was kept at a constant 37°C. The composition of the physiological solution was (mM): 135 NaCl, 5 KCl, 2.5 CaCl2, 1 MgSO4, 1 NaH2PO4, 15 NaHCO3 and 11 glucose, with an adjusted pH of 7.35–7.45 with HCl and sodium bicarbonate.

Diaphragm muscle was placed in between parallel platinum electrodes situated ~4 mm apart. The muscles were stimulated using a pulse width of 0.2 ms. The voltage was increased progressively until there was no further augmentation of contraction, and an additional 25% was added to this value (i.e. supramaximal stimulation; Seow & Stephens, 1988; Ameredes et al. 2000; Machiels et al. 2001). The contractile performance was measured with a dual-mode servo-controlled force transducer (model 300B, Aurora Scientific, Ontario, Canada). This force transducer measured length and force separately and was able to hold the force constant while the displacement was measured. Isometric force was determined at optimal length (Lo) during twitch contractions as well as 25, 50 or 75 Hz trains. Afterloads selected for use during isotonic contractions were set at specific percentages of these values, which were obtained separately for each muscle sample studied. Muscle fatigue during isotonic contractions was evaluated in a separate set of animals by repeatedly stimulating the muscle strip with 50 Hz trains and the afterload set at 20%. The muscle was stimulated every 3 s with a train duration of 333 ms for fatigue studies. The relatively long time in between trains was chosen based on a need for sufficient time in between contractions to allow any myotonic activity to end and relaxation to be complete prior to the onset of the next contraction. The choice of the 25, 50 and 75 Hz stimulation frequencies was based on the following considerations. First, a range of degrees of contractile fusion are represented. Second, previous studies of diaphragm isotonic contractile performance have used values within the 25–75 Hz range (Watchko et al. 1997; Zhan et al. 1998; Attal et al. 2000); the choice of similar frequencies allows the present data to be compared more easily with previous studies. Third, diaphragm motor unit firing frequencies in the rat range from 34 to 76 Hz with a mean of 56 Hz during resting breathing (Kong & Berger, 1986); we are not aware of similar data in the mouse, but suspect mouse firing frequencies are closer to that of the rat than that of other species for which diaphragm motor unit firing frequencies are known (e.g. cat).

Data were relayed to a computer using the data acquisition and analysis program Dynamic Muscle Control (Aurora Scientific Inc., Ontario, Canada). Isotonic contractile performance was evaluated by measuring the total amount of shortening, shortening velocity, work and power. The total amount of shortening was quantified based on the total length change during the contraction (Fig. 1). The velocity of shortening was calculated from the early portion of the contraction (first 5 ms), which corresponds to the time during which the velocity is close to or at its maximal value. Work was calculated as the product of the isotonic afterload and the total amount of shortening (and thus is not affected by the time course over which the shortening occurred). Power was calculated as the product of the isotonic afterload and shortening velocity measured during the early portion of the contraction. All values presented are means ± 1 S.E.M. Statistical analysis between two groups was performed using Student's unpaired t tests. Comparisons of muscle contractile parameters as a function of load and during fatigue were analysed with two-way repeated measures analysis of variance (RMANOVA), which was followed with the Neuman–Keuls test in the event of statistical significance. P values of < 0.05 (two-tailed) were considered to indicate statistical significance.


Figure 1
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  		Figure 1.  Example of muscle shortening during isotonic contraction, depicting methods for quantifying total shortening and velocity of shortening
Note that velocity of shortening was measured from the early portion of the contraction (5 ms), which is when values were at or near their maximum.

 

   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The diaphragm of CLC-1-deficient animals had impaired isometric force generation over a range of stimulation frequencies (Table 1). This is in agreement with data obtained from similar animals in a recent study which focused on the isometric contraction properties of myotonic muscle (van Lunteren et al. 2007).


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  		Table 1.  Values for isometric force as a function of stimulation frequency for normal and CLC-1-deficient muscle
 
The velocity of muscle shortening as a function of load and stimulation frequency is shown in Fig. 2. As expected, there was a reciprocal relationship between velocity and load. When plotted as a function of relative load, the velocity of shortening was reduced in CLC-1-deficient muscle during 50 Hz stimulation, especially at the lower loads, but was not significantly impaired during 25 and 75 Hz stimulation (left panels of Fig. 2). However, a plot of the data as a function of absolute load demonstrated impaired shortening velocity at most loads and frequencies (right panels of Fig. 2). Values for power of normal and CLC-1-deficient diaphragm at various loads and stimulation frequencies are depicted in Fig. 3. Compared with normal muscle, power of CLC-1 chloride channel-deficient muscle was considerably reduced across many loads and at all stimulation frequencies.


Figure 2
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  		Figure 2.  Shortening velocity of normal and CLC-1 chloride channel-deficient muscle as a function of load and stimulation frequency
For left panels, loads are expressed relative to maximal load for each muscle, whereas for right panels loads are expressed in absolute values. In left panels, *P < 0.05 between normal and diseased muscle (two-way RMANOVA followed by the Neuman–Keuls test).

 

Figure 3
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  		Figure 3.  Impaired power of CLC-1 chloride channel-deficient muscle as a function of load and stimulation frequency
For left panels, loads are expressed relative to maximal load for each muscle, whereas for right panels loads are expressed in absolute values. In left panels, *P < 0.05 between normal and diseased muscle (two-way RMANOVA followed by the Neuman–Keuls test).

 
The total amount of muscle shortening as a function of load and stimulation frequency is shown in Fig. 4. When plotted as a function of relative load, at 25 and 75 Hz stimulation the total shortening was similar for normal and CLC-1-deficient muscle, but at 50 Hz it was less than normal for CLC-1-deficient muscle. In contrast, a plot of the shortening values as a function of absolute load demonstrated substantial divergence between normal and myotonic data over a greater range of stimulation frequencies. Values for work of normal and CLC-1-deficient diaphragm are depicted in Fig. 5. Work was substantially lower for myotonic than normal muscle at several combinations of loads and stimulation frequencies.


Figure 4
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  		Figure 4.  Extent of total isotonic shortening of normal and CLC-1 chloride channel-deficient muscle as a function of load and stimulation frequency
For left panels, loads are expressed relative to maximal load for each muscle, whereas for right panels loads are expressed in absolute values. In left panels, *P < 0.05 between normal and diseased muscle (two-way RMANOVA followed by the Neuman–Keuls test).

 

Figure 5
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  		Figure 5.  Impaired work of CLC-1 chloride channel-deficient muscle as a function of load and stimulation frequency
For left panels, loads are expressed relative to maximal load for each muscle, whereas for right panels loads are expressed in absolute values. In left panels, *P < 0.05 between normal and diseased muscle (two-way RMANOVA followed by the Neuman–Keuls test).

 
The maximal unloaded shortening velocity (Vmax) was calculated, based on Hill's equation (Hill, 1938; Hatcher & Luff, 1985), using two approaches. The first used the group mean data as a function of absolute load from the right panels of Fig. 2 and fitted this to Hill's equation. The resultant best-fit curves are depicted in Fig. 6A and the calculated values for Vmax are depicted in Fig. 6Ba. The second approach calculated Vmax separately for each muscle sample, and then averaged values for the group of muscles (Fig. 6Bb). The best-fit curves were displaced downward and leftward for myotonic muscle during 50 and 75 Hz stimulation (Fig. 6A). However, calculated Vmax values were not significantly different for myotonic compared with normal muscle at any stimulation frequency (range of P values, 0.28–0.49; Fig. 6B).


Figure 6
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  		Figure 6.  Data for the load–shortening velocity relationship fit to Hill's equation to estimate maximal unloaded shortening velocity (Vmax)
A depicts best-fit curves for mean data presented in right panels of Fig. 2. B depicts calculated Vmax values. Ba is based on the curves shown in A. Bb is based on first determining Vmax values separately for each muscle, and then averaging Vmax values across each of the individual muscle samples.

 
To ensure that all muscle samples had sufficient time to return to initial length in between contractions, fatigue studies were performed at a rate of one contraction every 3 s.

During repetitive stimulation, the degree of myotonia generally decreased over time (Fig. 7), consistent with the ‘warm-up’ phenomenon that is well described in the myotonias (Ptacek et al. 1993).


Figure 7
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  		Figure 7.  Changes in muscle relaxation during repetitive stimulation
A, example of muscle isotonic shortening during repetitive train stimulation. Myotonic muscle demonstrates considerably slowed relaxation compared with normal. However, the extent of slowed relaxation is attenuated over the course of repeated contractions. B, changes in relaxation time during the course of repetitive stimulation depicted for diaphragm muscle from five different CLC-1-deficient myotonic animals (indicated by different symbols) during repetitive 50 Hz train stimulation. (Relaxation time for normal muscle did not shorten during repetitive stimulation; data not shown.)

 
In response to fatigue-inducing 50 Hz stimulation at 20% load, there was a transient increase followed a progressive reduction in power and velocity of shortening (Fig. 8). Work and shortening, in contrast, decreased progressively over time for both normal and myotonic muscle (Fig. 9). Power, work, shortening velocity and total shortening were significantly lower for myotonic than normal muscle during fatigue-inducing stimulation. Differences between healthy and diseased muscle were more prominent early than late during the fatigue test, and were generally greater for work and power than for shortening velocity. For power and shortening velocity, the pattern of changes over time was similar for normal and myotonic muscle (Fig. 8). In contrast, for work and shortening, declines were much steeper at the onset of stimulation for CLC-1-deficient than normal muscle (Fig. 9).


Figure 8
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  		Figure 8.  Changes in power and shortening velocity over time during fatigue-inducing repetitive stimulation
*P < 0.05 between normal and diseased muscle (two-way RMANOVA followed by the Neuman–Keuls test).

 

Figure 9
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  		Figure 9.  Changes in work and shortening over time during fatigue-inducing repetitive stimulation
*P < 0.05 between normal and diseased muscle (two-way RMANOVA followed by the Neuman–Keuls test).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The present study confirmed the presence of myotonia in diaphragm muscle of the CLC-1 chloride channel-deficient mouse, as evidenced by a prolonged period of time required for relaxation to be complete. The focus of the present study, however, was on the contractile phase of the contraction–relaxation cycle, and found the following. First, there was a considerable impairment of diaphragm muscle power and work with murine CLC-1 chloride channel deficiency, which was present across a range of loads and stimulation frequencies. Second, CLC-1 chloride channel-deficient diaphragm demonstrated impaired shortening and velocity of shortening, in particular when plotted as a function of absolute rather than relative load. Third, during fatigue-inducing stimulation, power, work and shortening velocity were significantly impaired in myotonic muscle. Finally, CLC-1 chloride channel-deficient diaphragm had a much faster initial decline in work and shortening than normal muscle at the onset of repetitive stimulation.

Only a few studies have examined the isometric contractile properties of skeletal muscle in myotonic mice. Entrikin et al. (1987) studied extensor digitorum muscle in the mto/mto mouse. They noted that peak tetanic tension was reduced from a control value of 13g to 6.7g in diseased animals, but owing to the smaller muscle size in myotonic mice, peak tension per unit muscle weight was not impaired (1.8 versus 1.7 g tension (g muscle weight)–1). No information was provided about whether the muscles from diseased animals were shorter, less wide, or both; thus it is unclear whether the force normalized for cross-sectional area was impaired. The rate of tension development in myotonic muscle was reduced from 484 to 160 g s–1, which is out of proportion to both the reduction in muscle weight and peak isometric force. In addition, relaxation rate was reduced from 1096 to 49 g s–1 and the half-relaxation time increased from 20 to 549 ms in the muscle from myotonic animals.

A year later, Reininghaus et al. (1988) reported isometric contractile properties of ADR mice. For the anterior tibialis muscle, twitch tension was not impaired in 10- to 20-day-old mice, but was reduced to 60% of control values in 60-day-old animals (at which time, muscle mass averaged 70% of control values). As in the study of Entrikin et al. (1987), muscle cross-sectional area was not used to normalize force values. Up to the age of 60 days, isometric contraction and relaxation times during twitch contractions were similar in normal and diseased animals. In contrast, at ages of 80 and 95 days, both values were longer than normal in ADR mice, with half-relaxation time ranging from approximately 5–7 ms in normal and 9–11 ms in diseased muscle. In contrast, during contractions elicited at frequencies exceeding 10 Hz, after-contractions were prominent even at young ages (10 days postnatal), and were found in both anterior tibialis and soleus muscle. The longevity of the after-contractions was not quantified, but representative force traces demonstrated that myotonia persisted much longer than the duration of the antecedent contraction.

The present study agrees with the above two studies, as well as with original descriptions of these animals, in several respects. Our data on reduced power of CLC-1-deficient muscle agree with Watkins & Watts (1984) that the animals are weak. Both Entrikin et al. (1987) and Reininghaus et al. (1988) report reduced isometric forces, although it is not clear whether force per unit cross-sectional area was reduced. Of note, the extent of force reduction in myotonic muscle reported by Entrikin et al. (1987) was similar to that found in {alpha}2-laminin-deficient dy/dy dystrophic mice in the same study. This suggests that myotonic muscle indeed had impaired force production in their study, since muscles from dy/dy dystrophic mice are well documented to have severely impaired isometric force (Hayes & Williams, 1998; van Lunteren & Moyer, 2002). Recent data from our laboratory support the point of view that isometric contractile performance is diminished in myotonic mice (van Lunteren et al. 2007). The present study extends this by demonstrating that isotonic contractile performance is impaired as well.

With regard to the mechanisms underlying the isotonic contractile changes, myotonic muscle has no or at most minimal evidence of fibre atrophy, necrosis, regeneration or inflammation (Heller et al. 1982; Reininghaus et al. 1988), in contrast to myotonic dystrophy and the muscular dystrophies, in which these changes are extensive. A number of studies have demonstrated the absence or near absence of type II glycolytic fibres in the myotonias. Reininghaus et al. (1988) studied anterior tibialis muscle in the mouse model of myotonia congenita. They found that muscle from normal animals was comprised of 44–50% type II glycolytic fibres, whereas that from myotonic mice had no type II glycolytic fibres; the proportion of type I fibres did not differ between normal and myotonic muscle. An absence of type II glycolytic fibres was also found in several trunk muscles. Diaphragm and intercostal muscles had reduced but not absent type II glycolytic fibres (although no quantitative data were provided). More recently, Agbulut et al. (2004) measured myosin heavy chain isoforms in myotonic mice. The tibialis anterior muscle of myotonic mice had a shift in myosin heavy chain expression from IIB to IIA, along with modest reduction of IIX, compared with normal mice. The diaphragm of normal animals had all four adult myosin heavy chains (IIX and IIA > I > IIB), whereas in ADR myotonic mice myosin heavy chain IIB was absent, IIX was increased, IIA remained relatively unchanged and I was decreased. Goblet & Whalen (1995) confirmed reduced IIB myosin with increased amounts of other myosin isoforms at a mRNA level and, furthermore, linked this to abnormal expression of myogenic regulatory factors (MyoD and myogenin). Human myotonic muscle is similar to rat myotonic muscle in having reduced proportions of type IIB fibres (Heene et al. 1986). Regarding structural and biochemical properties other than those involving fibre types and myosin isoforms, Stuhlfauth et al. (1984) reported downregulation of the calcium-binding protein parvalbumin, which has been confirmed by others.

The marked slowing of relaxation found in the present study is undoubtedly due to the altered chloride conductance. Regarding mechanisms underlying the changes in performance during the contraction phase of the contraction–relaxation cycle, a few things need to be considered. First, the myosin isoform changes reported by Agbulut et al. (2004) are somewhat complex. Type IIB was replaced by type IIX, which should make the muscle ‘slower’ (Bottinelli et al. 1994) and, at the same time, type I was replaced by type IIX, which should make the muscle ‘faster’. The net effect on the muscle as a whole depends on the magnitude of these changes, as well as the functional impact of a IIB to IIX change compared with a I to IIX change. A further complication is that among fibres of each of the above four types (I, IIA, IIX and IIB) there is a range of maximal shortening velocities (Bottinelli et al. 1994), so that the location within that range of the normal and diseased fibres for each of the four fibre types will also have a large impact on the overall contractile properties of the whole muscle. Second, there may be changes in absolute amounts of myosin in myotonic muscle in addition to shifts in myosin isoform percentages, and these reductions can impact contractile properties. That is, the study of Agbulut et al. (2004) provides us with data on myosin isoform proportions, but does not tell us whether there are changes in total myosin content in myotonic muscle relative to normal muscle. Thus, it is unclear whether loss of type IIB and I myosins leads to compensatory increases in type IIX myosin, or whether there is loss of type IIB and I myosins without any increase in IIX so that the relative proportion of IIX myosin is higher but the total amount of myosin is now lower. A reduction in total myosin content can easily explain decrements in force and shortening, irrespective of changes in relative proportions of myosin isoforms. Third, there may be many other changes present in myotonic muscle, in addition to shifts in myosin changes, that can impact on contractile properties. This includes amounts of other contractile proteins and levels of enzymes involved in cellular energetics, none of which, to the best of our knowledge, has been measured in myotonic muscle.

During fatigue testing, in some instances there were temporal differences between changes in shortening and velocity of shortening. In particular, the velocity of shortening was well maintained yet total shortening declined rapidly for myotonic muscle. Thus, the initial rate of muscle shortening is relatively well preserved, but it cannot be maintained for a very long period of time during the course of the contraction. Hence, shortening during the later part of the contraction is impaired, so that total shortening decreases. Presumably, this is similar to the phenomenon of intratrain fatigue that is apparent during studies of isometric contractions, in which isometric force reaches a peak value but then drops during the remainder of the train (e.g. see van Lunteren & Moyer, 1996).

In the present study, we used a train duration of 333 ms, which may be slightly longer than the normal duration of contraction in the intact animal. This should not have affected values for velocity of shortening (or power), in that velocity was measured during the early portion of the contraction. The effect on shortening (and work) should be small, in that most of the shortening occurred during the early portion of the contraction. Another issue is that the 25 and 75 Hz data for work (Fig. 3) did not reach statistical significance despite relative large numerical differences between myotonic and normal data values, although there was a statistical trend in this direction. This is most probably due to the higher degree of variability within each set of data (note, in particular, large S.E.M. bars for 75 Hz data), since the statistical approaches incorporate both differences between the mean values of the two groups as well as the degree of variability within each group.

The loads against which the muscles contracted were set at specific percentages of the maximal isometric force that each muscle sample could produce, consistent with standard methodology for isotonic contractile studies (Seow & Stephens, 1988; Watchko et al. 1997; Kelly & McCarter, 1993; Machiels et al. 2001; Vedsted et al. 2003). As a result, there is a bias against muscles with higher intrinsic force-generating capacity during testing of fatigue resistance (in this study, the normal muscles), in that the loads they face are higher in absolute terms despite being matched with regards to the percentage of the maximal load. Despite this bias, wild-type muscle had better contractile values than myotonic muscle during the course of fatigue-inducing stimulation in the present study, so that the net effect is an underestimation of the extent to which the myotonic muscle was impaired.

In conclusion, genetic CLC-1 chloride channel deficiency alters diaphragm muscle isotonic contractile performance in manners which go well beyond the slowing of relaxation. Power, work, velocity of shortening and extent of shortening were all reduced for several or many combinations of load and stimulation frequency in myotonic compared with normal mice. Furthermore, these deficits persisted during fatigue-inducing repetitive stimulation. These findings indicate that the fibre subtype and myosin isoform shifts previously described in myotonic muscle (Heene et al. 1986; Reininghaus et al. 1988; Agbulut et al. 2004) have important functional consequences during the contractile phase of isotonic contractions.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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   Acknowledgements
 
This study was supported in part by NIH Grant HL-70697 as well as grants from the Department of Veterans Affairs.




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