Improvement of diaphragm and limb muscle isotonic contractile performance by K+ channel blockade.

The K+ channel blocking aminopyridines greatly improve skeletal muscle isometric contractile performance during low to intermediate stimulation frequencies, making them potentially useful as inotropic agents for functional neuromuscular stimulation applications. Most restorative applications involve muscle shortening; however, previous studies on the effects of aminopyridines have involved muscle being held at constant length. Isotonic contractions differ substantially from isometric contractions at a cellular level with regards to factors such as cross-bridge formation and energetic requirements. The present study tested effects of 3,4-diaminopyridine (DAP) on isotonic contractile performance of diaphragm, extensor digitorum longus (EDL) and soleus muscles from rats. During contractions elicited during 20 Hz stimulation, DAP improved work over a range of loads for all three muscles. In contrast, peak power was augmented for the diaphragm and EDL but not the soleus. Maintenance of increased work and peak power was tested during repetitive fatigue-inducing stimulation using a single load of 40% and a stimulation frequency of 20 Hz. Work and peak power of both diaphragm and EDL were augmented by DAP for considerable periods of time, whereas that of soleus muscle was not affected significantly. These results demonstrate that DAP greatly improves both work and peak power of the diaphragm and EDL muscle during isotonic contractions, which combined with previous data on isometric contractions indicates that this agent is suitable for enhancing muscle performance during a range of contractile modalities.

Inotropic effects of the K+ channel blocker 3,4-diaminopyridine on fatigued diaphragm muscle.

K(+) channels play important roles in skeletal muscle contraction by regulating action potential duration. Blocking these channels, for example with 3,4-diaminopyridine (DAP), augments muscle force considerably, and these force increases are maintained well during fatigue-inducing contractions. The present study tested the hypothesis that K(+) channel blockade also improves force of previously fatigued muscle. Rat diaphragm underwent fatigue-inducing stimulation in vitro with four different stimulation protocols consisting of 20 Hz vs. 50 Hz trains and 1 min vs. 4 min stimulation durations. DAP administered at the onset of the recovery period produced significant force increases irrespective of the amount of antecedent force loss. These force gains considerably exceeded those resulting from normal force recovery in untreated muscle. Furthermore contraction time was prolonged by DAP in all cases, and half-relaxation time was prolonged by DAP in most cases. Several differences were found compared with previous studies of DAP in fresh muscle, including smaller magnitude and slower time course of force increases. Intracellular electrophysiological recordings found smaller effects of DAP on action potential overshoot and time-depolarization integral in previously stimulated compared with fresh muscle. These data indicate that K(+) channel blockade does indeed increase force of fatigued diaphragm, but to an attenuated extent relative to its effects on non-fatigued muscle, which can be explained on the basis of electrophysiological findings. Nonetheless DAP-induced force increases were usually sufficient to restore force to values present prior to the onset of fatigue-inducing stimulation.

Genetic CLC-1 chloride channel deficiency modifies diaphragm muscle isometric contractile properties.

Genetic deficiency of the muscle chloride channel CLC-1 leads to myotonia congenita in humans as well as myotonia in mice and goats. The hallmark of myotonia is delayed muscle relaxation due to persistent electrical discharges in the muscle. The present study tested the hypothesis that performance of CLC-1 deficient diaphragm muscle is also altered during the contractile phase of the contraction-relaxation cycle. Diaphragm of CLC-1 deficient and wild type mice underwent in vitro isometric contractility testing. Myotonia was easily demonstrable during contractions elicited by train stimulation, but was not seen during twitch stimulation or during train stimulation preceded by a series of twitch stimulations. Twitch force was reduced from 16.7+/-2.5 N/cm(2) in normal muscle to 7.2+/-1.9 N/cm(2) in CLC-1 deficient muscle (P<0.002). Isometric twitch contraction time was shortened from 19.6+/-0.9 to 15.7+/-1.0 ms (P<0.002). During repetitive 25 Hz stimulation, force/area was lower for diseased than normal muscle, whereas force as a percent of initial values declined at a faster rate for normal than diseased muscle. The latter could be accounted for by a rightward shift in the force-frequency relationship of CLC-1 deficient relative to normal muscle, as use of stimulation frequencies which elicited comparable force levels as a percentage of maximum 100 Hz tetanic force led to similar rates of fatigue. These findings indicate that genetic CLC-1 deficiency not only affects muscle relaxation (myotonia) but also modulates diaphragm performance during the contractile phase of the contraction-relaxation cycle.

Inotropic effects of the K+ channel blocker 3,4-diaminopyridine: differential responses of rat soleus and extensor digitorum longus.

The K+ channel blocker 3,4-diaminopyrindine (DAP) increases diaphragm force, use of which could potentially improve muscle performance during functional neuromuscular stimulation. To determine the extent of hindlimb muscle force augmentation, and delineate whether DAP effects vary in muscles comprised of mainly slow versus fast fibers, rat soleus, extensor digitorum longus (EDL) and diaphragm muscle samples were studied in vitro. DAP increased force of all three muscles, but at high concentrations the force increases were transient and were followed by declines in force below baseline. The maximum DAP-induced twitch force increase was smaller for soleus (38 +/-7%) than both EDL (94+/-12%) (P < 0.05) and diaphragm (93+/-13%) (P < 0.01). During fatigue-inducing 20 Hz stimulation (tested at an intermediate DAP concentration), force of soleus muscle remained significantly elevated by DAP for the entire testing period, force of DAP-treated EDL muscle rapidly declined to values in untreated muscle, and force of DAP-treated diaphragm had an intermediate force-time profile. Muscles varied in extent to which isometric contractile kinetics were altered by DAP. Thus, the K+ channel blocker DAP improves contractile performance of limb muscles, but the profile of improvement is distinct between the soleus and EDL muscles.

Reduced amount and disrupted temporal pattern of spontaneous exercise in diabetic rats.

INTRODUCTION/PURPOSE: The beneficial effects of exercise for subjects with diabetes or prediabetic states are well established. However, the converse, that is, the effect of diabetes on spontaneous exercise performance, is not as well defined. Mice with mdx muscular dystrophy not only reduce total spontaneous running distance, but also decrease the duration of periods during which they are active, suggesting a defect in endurance. Studies tested the hypothesis that Type I diabetes causes similar changes in spontaneous exercise performance. METHODS: Wistar rats received streptozotocin to produce a model of Type I diabetes or buffer alone, and had access to running wheels for the next 8 wk. RESULTS: Diabetic rats had elevated serum glucose levels (689 +/- 85 vs 270 +/- 21 mg x dL(-1), P = 0.0003) but normal serum bicarbonate levels. After 8 wk, diabetic rats were running for considerably lower distances than normal animals (daily distance 182 +/- 58 vs 4981 +/- 1373 m, P = 0.006). Furthermore, the average consecutive running time was much shorter in diabetic than normal rats (16 +/- 1 vs 40 +/- 6 min, P = 0.004). Differences in running behavior between diabetic and normal mice were absent early after injection of streptozotocin, but were fully established by week 4 for both total distance and consecutive running times. CONCLUSION: Severe untreated Type I diabetes in rats reduces spontaneous exercise in a manner similar to that seen in mdx mouse muscular dystrophy, with reduced running distance and consecutive running times.

Impaired Wheel Running Exercise in CLC-1 Chloride Channel-Deficient Myotonic Mice.

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.

Long-lasting in vivo inotropic effects of the K(+) channel blocker 3,4-diaminopyridine during fatigue-inducing stimulation.

Blocking K(+) channels with aminopyridines enhances muscle contractile performance in vitro, but the improvements are relatively short-lasting during fatigue-inducing stimulation. We hypothesized that in vivo inotropic actions persist over long periods of fatigue-inducing stimulation. The effects of 3,4-diaminopyridine (DAP) were evaluated for rat extensor digitorum longus (EDL) muscle. DAP increased twitch force by 105%. There was a significant leftward shift in the force-frequency relationship, with force values being increased at frequencies up to and including 20 HZ. During repetitive fatigue-inducing 20-HZ stimulation, DAP-induced force increases were large and persisted significantly for at least 30 minutes. Thus, DAP substantially improves contractile performance of EDL muscle in vivo for much longer periods during fatigue-inducing contractions than in vitro. These data provide support for a potential role for aminopyridines as inotropic agents in applications such as functional electrical stimulation, in which low to medium stimulation frequencies are typically utilized.

Isotonic contractile impairment due to genetic CLC-1 chloride channel deficiency in myotonic mouse diaphragm muscle.

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-Clcn1(adr-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.

Isotonic fatigue in laminin alpha2-deficient dy/dy dystrophic mouse diaphragm.

Laminin alpha2 deficiency causes approximately 50% of human congenital muscular dystrophies. Muscle in the corresponding dy/dy mouse model has reduced force but increased fatigue resistance during isometric contractions. To determine whether a similar pattern of alterations is present during isotonic contractions, dy/dy diaphragm was studied in vitro. During 20% load, dystrophic diaphragm had significantly reduced shortening, shortening velocity, work and power deficits, which persisted during the fatigue-inducing stimulation. In contrast, during 40% load, isotonic contractile performance of diseased muscle was impaired only mildly and only for some contractile parameters. At both loads, rate of isotonic fatigue when expressed relative to initial contractile values was similar for dystrophic and normal muscle, or in some instances slightly higher for dystrophic muscle. Therefore, fatigue resistance is considerably impaired during isotonic contractions relative to that reported previously for isometric contractions. This has important implications for increased susceptibility to respiratory failure in laminin alpha2-deficient muscular dystrophy.