Hyperammonemia results in reduced muscle function independent of muscle mass.

The mechanism of the nearly universal decreased muscle strength in cirrhosis is not known. We evaluated whether hyperammonemia in cirrhosis causes contractile dysfunction independent of reduced skeletal muscle mass. Maximum grip strength and muscle fatigue response were determined in cirrhotic patients and controls. Blood and muscle ammonia concentrations and grip strength normalized to lean body mass were measured in the portacaval anastomosis (PCA) and sham-operated pair-fed control rats (n = 5 each). Ex vivo contractile studies in the soleus muscle from a separate group of Sprague-Dawley rats (n = 7) were performed. Skeletal muscle force of contraction, rate of force development, and rate of relaxation were measured. Muscles were also subjected to a series of pulse trains at a range of stimulation frequencies from 20 to 110 Hz. Cirrhotic patients had lower maximum grip strength and greater muscle fatigue than control subjects. PCA rats had a 52.7 ± 13% lower normalized grip strength compared with control rats, and grip strength correlated with the blood and muscle ammonia concentrations (r(2) = 0.82). In ex vivo muscle preparations following a single pulse, the maximal force, rate of force development, and rate of relaxation were 12.1 ± 3.5 g vs. 6.2 ± 2.1 g; 398.2 ± 100.4 g/s vs. 163.8 ± 97.4 g/s; -101.2 ± 22.2 g/s vs. -33.6 ± 22.3 g/s in ammonia-treated compared with control muscle preparation, respectively (P < 0.001 for all comparisons). Tetanic force, rate of force development, and rate of relaxation were depressed across a range of stimulation from 20 to 110 Hz. These data provide the first direct evidence that hyperammonemia impairs skeletal muscle strength and increased muscle fatigue and identifies a potential therapeutic target in cirrhotic patients.

Alterations in lung gene expression in streptozotocin-induced diabetic rats.

BACKGROUND: Diabetes profoundly affects gene expression in organs such as heart, skeletal muscle, kidney and liver, with areas of perturbation including carbohydrate and lipid metabolism, oxidative stress, and protein ubiquitination. Type 1 diabetes impairs lung function, but whether gene expression alterations in the lung parallel those of other tissue types is largely unexplored. METHODS: Lung from a rat model of diabetes mellitus induced by streptozotocin was subjected to gene expression microarray analysis. RESULTS: Glucose levels were 67 and 260 mg/dl (p < 0.001) in control and diabetic rats, respectively. There were 46 genes with at least ± 1.5-fold significantly altered expression (19 increases, 27 decreases). Gene ontology groups with significant over-representation among genes with altered expression included apoptosis, response to stress (p = 0.03), regulation of protein kinase activity (p = 0.04), ion transporter activity (p = 0.01) and collagen (p = 0.01). All genes assigned to the apoptosis and response to stress groups had increased expression whereas all genes assigned to the collagen group had decreased expression. In contrast, the protein kinase activity and ion transporter activity groups had genes with both increased and decreased expression. CONCLUSIONS: Gene expression in the lung is affected by type 1 diabetes in several specific areas, including apoptosis. However, the lung is resistant to changes in gene expression related to lipid and carbohydrate metabolism and oxidative stress that occur in other tissue types such as heart, skeletal muscle and kidney.

Gene expression of sternohyoid and diaphragm muscles in type 2 diabetic rats.

BACKGROUND: Type 2 diabetes differs from type 1 diabetes in its pathogenesis. Type 1 diabetic diaphragm has altered gene expression which includes lipid and carbohydrate metabolism, ubiquitination and oxidoreductase activity. The objectives of the present study were to assess respiratory muscle gene expression changes in type 2 diabetes and to determine whether they are greater for the diaphragm than an upper airway muscle. METHODS: Diaphragm and sternohyoid muscle from Zucker diabetic fatty (ZDF) rats were analyzed with Affymetrix gene expression arrays. RESULTS: The two muscles had 97 and 102 genes, respectively, with at least ± 1.5-fold significantly changed expression with diabetes, and these were assigned to gene ontology groups based on over-representation analysis. Several significantly changed groups were common to both muscles, including lipid metabolism, carbohydrate metabolism, muscle contraction, ion transport and collagen, although the number of genes and the specific genes involved differed considerably for the two muscles. In both muscles there was a shift in metabolism gene expression from carbohydrate metabolism toward lipid metabolism, but the shift was greater and involved more genes in diabetic diaphragm than diabetic sternohyoid muscle. Groups present in only diaphragm were blood circulation and oxidoreductase activity. Groups present in only sternohyoid were immune & inflammation and response to stress & wounding, with complement genes being a prominent component. CONCLUSION: Type 2 diabetes-induced gene expression changes in respiratory muscles has both similarities and differences relative to previous data on type 1 diabetes gene expression. Furthermore, the diabetic alterations in gene expression differ between diaphragm and sternohyoid.

The Effects of K(+) Channel Blockade on Eccentric and Isotonic Twitch and Fatiguing Contractions in situ.

K(+) channel blockers like 3,4-diaminopyridine (DAP) can double isometric muscle force. Functional movements require more complex concentric and eccentric contractions, however the effects of K(+) channel blockade on these types of contractions in situ are unknown. Extensor digitorum longus (EDL) muscles were stimulated in situ with and without DAP in anesthetized rats and fatigability was addressed using a series of either concentric or eccentric contractions. During isotonic protocols (5-100% load), DAP significantly shifted shortening- and maximum shortening velocity-load curves upward and to the right and increased power and work. Maximum shortening, maximum shortening velocity, and power doubled while work increased by ∼250% during isotonic contraction at 50% load. During isotonic fatigue, DAP significantly augmented maximum shortening, work, shortening velocity, and power. During constant velocity eccentric protocols (2-12 mm/s), DAP increased muscle force during eccentric contractions at 6, 8, 10, and 12 mm/s. During eccentric contraction at a constant velocity of 6 mm/s while varying the stimulation frequency, DAP significantly increased muscle force during 20, 40, and 70 Hz. The effects of DAP on muscle contractile performance during eccentric fatigue varied with level of fatigue. DAP-induced contractile increases during isotonic contractions were similar to those produced during previously studied isometric contractions, while the DAP effect during eccentric contractions was more modest. These findings are especially important in attempting to optimize functional electrical stimulation parameters for spinal cord injury patients while also preventing rapid fatigue of those muscles.

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.

Expression of a dominant negative CELF protein in vivo leads to altered muscle organization, fiber size, and subtype.

BACKGROUND: CUG-BP and ETR-3-like factor (CELF) proteins regulate tissue- and developmental stage-specific alternative splicing in striated muscle. We previously demonstrated that heart muscle-specific expression of a nuclear dominant negative CELF protein in transgenic mice (MHC-CELFΔ) effectively disrupts endogenous CELF activity in the heart in vivo, resulting in impaired cardiac function. In this study, transgenic mice that express the dominant negative protein under a skeletal muscle-specific promoter (Myo-CELFΔ) were generated to investigate the role of CELF-mediated alternative splicing programs in normal skeletal muscle. METHODOLOGY/PRINCIPAL FINDINGS: Myo-CELFΔ mice exhibit modest changes in CELF-mediated alternative splicing in skeletal muscle, accompanied by a reduction of endomysial and perimysial spaces, an increase in fiber size variability, and an increase in slow twitch muscle fibers. Weight gain and mean body weight, total number of muscle fibers, and overall muscle strength were not affected. CONCLUSIONS/SIGNIFICANCE: Although these findings demonstrate that CELF activity contributes to the normal alternative splicing of a subset of muscle transcripts in vivo, the mildness of the effects in Myo-CELFΔ muscles compared to those in MHC-CELFΔ hearts suggests CELF activity may be less determinative for alternative splicing in skeletal muscle than in heart muscle. Nonetheless, even these small changes in CELF-mediated splicing regulation were sufficient to alter muscle organization and muscle fiber properties affected in myotonic dystrophy. This lends further evidence to the hypothesis that dysregulation of CELF-mediated alternative splicing programs may be responsible for the disruption of these properties during muscle pathogenesis.

Differential susceptibility of muscles to myotonia and force impairment in a mouse model of myotonic dystrophy.

INTRODUCTION: Myotonic dystrophy, or dystrophia myotonica (DM), is characterized by prominent muscle wasting and weakness as well as delayed muscle relaxation resulting from persistent electrical discharges. METHODS: We hypothesized heterogeneity among muscles in degree of weakness and myotonia in an expanded [(CUG)(250)] repeats transgenic (HSA(LR)) mouse DM model. Muscle contraction was compared among diaphragm, extensor digitorum longus (EDL), and soleus muscles. RESULTS: Myotonia was found only in EDL, as manifested by longer late-relaxation time and elevated myotonic index. EDL, but not the other two muscles, had impaired force over a wide range of stimulation frequencies. During fatigue-inducing stimulation, DM EDL muscle force per cross-sectional area was significantly impaired during 25-Hz stimulation, whereas there were no differences in fatigue response for DM diaphragm or soleus. CONCLUSION: In an expanded repeats model of DM the EDL is more susceptible to myotonia and force impairment than muscles with lower proportions of fast-twitch fibers.

Fatigue-inducing stimulation resolves myotonia in a drug-induced model.

BACKGROUND: Slowed muscle relaxation is the contractile hallmark of myotonia congenita, a disease caused by genetic CLC-1 chloride channel deficiency, which improves with antecedent brief contractions ("warm-up phenomenon"). It is unclear to what extent the myotonia continues to dissipate during continued repetitive contractions and how this relates temporally to muscle fatigue. Diaphragm, EDL, and soleus muscles were examined in vitro during repetitive 20 Hz and 50 Hz train stimulation in a drug-induced (9-AC) rat myotonia model. RESULTS: At the onset of stimulation, 9-AC treated diaphragm and EDL muscle had markedly prolonged half relaxation and late relaxation times (range 147 to 884 ms, 894 to 1324 ms). Half relaxation and late relaxation times reached near-normal values over the 5-10 and 10-40 subsequent contractions, respectively. In both muscles myotonia declined faster during repetitive 50 Hz than 20 Hz stimulation, and much faster than the rate of force loss during fatigue at both frequencies. Soleus muscle was resistant to the myotonic effects of 9-AC. CONCLUSIONS: In a drug-induced model of mechanical myotonia, fatigue-inducing stimulation resolves the myotonia, which furthermore appears to be independent from the development of muscle fatigue.

Differential expression of lipid and carbohydrate metabolism genes in upper airway versus diaphragm muscle.

STUDY OBJECTIVES: Contractile properties of upper airway muscles influence upper airway patency, an issue of particular importance for subjects with obstructive sleep apnea. Expression of genes related to cellular energetics is, in turn, critical for the maintenance of contractile integrity over time during repetitive activation. We tested the hypothesis that sternohyoid has lower expression of genes related to lipid and carbohydrate energetic pathways than the diaphragm. METHODS: Sternohyoid and diaphragm from normal adult rats were examined with gene expression arrays. Analysis focused on genes belonging to Gene Ontology (GO) groups carbohydrate metabolism and lipid metabolism. RESULTS: There were 433 genes with at least +/- 2-fold significant differential expression between sternohyoid and diaphragm, of which 192 had sternohyoid > diaphragm and 241 had diaphragm > sternohyoid expression. Among genes with higher sternohyoid expression, there was over-representation of the GO group carbohydrate metabolism (P = 0.0053, n = 13 genes, range of differential expression 2.1- to 6.2-fold) but not lipid metabolism (P = 0.44). Conversely, among genes with higher diaphragm expression, there was over-representation of the GO group lipid metabolism (P = 0.0000065, n = 32 genes, range of differential expression 2.0- to 37.9-fold) but not carbohydrate metabolism (P = 0.23). Nineteen genes with diaphragm > sternohyoid expression were related to fatty acid metabolism (P = 0.000000058), in particular fatty acid beta oxidation and biosynthesis in the mitochondria. CONCLUSIONS: Sternohyoid has much lower gene expression than diaphragm for mitochondrial enzymes that participate in fatty acid oxidation and biosynthesis. This likely contributes to the lower fatigue resistance of pharyngeal upper airway muscles compared with the diaphragm.

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.

Gene expression profiling in the type 1 diabetes rat diaphragm.

BACKGROUND: Respiratory muscle contractile performance is impaired by diabetes, mechanisms of which included altered carbohydrate and lipid metabolism, oxidative stress and changes in membrane electrophysiology. The present study examined to what extent these cellular perturbations involve changes in gene expression. METHODOLOGY/PRINCIPAL FINDINGS: Diaphragm muscle from streptozotocin-diabetic rats was analyzed with Affymetrix gene expression arrays. Diaphragm from diabetic rats had 105 genes with at least +/-2-fold significantly changed expression (55 increased, 50 decreased), and these were assigned to gene ontology groups based on over-representation analysis using DAVID software. There was increased expression of genes involved in palmitoyl-CoA hydrolase activity (a component of lipid metabolism) (P = 0.037, n = 2 genes, fold change 4.2 to 27.5) and reduced expression of genes related to carbohydrate metabolism (P = 0.000061, n = 8 genes, fold change -2.0 to -8.5). Other gene ontology groups among upregulated genes were protein ubiquitination (P = 0.0053, n = 4, fold change 2.2 to 3.4), oxidoreductase activity (P = 0.024, n = 8, fold change 2.1 to 6.0), and morphogenesis (P = 0.012, n = 10, fold change 2.1 to 4.3). Other downregulated gene groups were extracellular region (including extracellular matrix and collagen) (P = 0.00032, n = 13, fold change -2.2 to -3.7) and organogenesis (P = 0.032, n = 7, fold change -2.1 to -3.7). Real-time PCR confirmed the directionality of changes in gene expression for 30 of 31 genes tested. CONCLUSIONS/SIGNIFICANCE: These data indicate that in diaphragm muscle type 1 diabetes increases expression of genes involved in lipid energetics, oxidative stress and protein ubiquitination, decreases expression of genes involved in carbohydrate metabolism, and has little effect on expression of ion channel genes. Reciprocal changes in expression of genes involved in carbohydrate and lipid metabolism may change the availability of energetic substrates and thereby directly modulate fatigue resistance, an important issue for a muscle like the diaphragm which needs to contract without rest for the entire lifetime of the organism.

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.

Contrast between cardiac left ventricle and diaphragm muscle in expression of genes involved in carbohydrate and lipid metabolism.

The heart and diaphragm both need appropriate metabolic machinery to ensure long-term energy supplies, as they must contract rhythmically without cessation for the entire lifetime of the organism to ensure homeostasis of oxygen and carbon dioxide exchange. However, their energy requirements differ due to disparities in mechanical loads. Understanding how these two muscles converge and diverge in their approaches to meeting their metabolic demands may suggest novel strategies for improving cardiac and skeletal muscle long-term performance in health and disease. To assess this at a transcriptional level, expression of genes involved in carbohydrate and lipid metabolism was assessed using microarrays in rats. There were 594 genes with >2-fold differential expression between left ventricle of the heart and diaphragm; 307 were expressed heart>diaphragm and 287 diaphragm>heart. Assignment to gene ontology groups revealed over-representation for "carbohydrate metabolism" (P=0.005, n=32 genes or 5.4% of all genes with differential expression) and "lipid metabolism" (P=0.0012, n=48 genes or 8.1% of all genes with differential expression). For carbohydrate there were 14 genes with heart>diaphragm and 18 genes with diaphragm>heart, and for lipid there were 30 genes with heart>diaphragm and 18 genes with diaphragm>heart. The magnitude of differential expression between heart and diaphragm ranged up to 30-fold for carbohydrate and up to 59-fold for lipid. Carbohydrate-related genes were almost all involved in energy metabolism (e.g. Pfkm, Pgm1, Pgam1, Pfkfb1, Pfkfb2), whereas lipid-related genes were involved in energetics as well as other cellular processes; for both groups this included genes involved in rate-limiting metabolic steps. Data thus indicate that diaphragm and heart have both shared and differential transcriptional strategies for ensuring long-term energy supplies, with a relative favoring of lipid metabolism in the heart and carbohydrate metabolism in the diaphragm.

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.

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.

Oxidoreductase, morphogenesis, extracellular matrix, and calcium ion-binding gene expression in streptozotocin-induced diabetic rat heart.

Diabetes has far-ranging effects on cardiac structure and function. Previous gene expression studies of the heart in animal models of type 1 diabetes concur that there is altered expression of genes involved in lipid and protein metabolism, but they diverge with regard to expression changes involving many other functional groups of genes of mechanistic importance in diabetes-induced cardiac dysfunction. To obtain additional information about these controversial areas, genome-wide expression was assessed using microarrays in left ventricle from streptozotocin-diabetic and normal rats. There were 261 genes with statistically significant altered expression of at least +/-1.5-fold, of which 124 were increased and 137 reduced by diabetes. Gene ontology assignment testing identified several statistical significantly overrepresented groups among genes with altered expression, which differed for increased compared with reduced expression. Relevant gene groups with increased expression by diabetes included lipid metabolism (P < 0.001, n = 13 genes, fold change 1.5 to 14.6) and oxidoreductase activity (P < 0.001, n = 17, fold change 1.5 to 4.6). Groups with reduced expression by diabetes included morphogenesis (P < 0.00001, n = 28, fold change -1.5 to -5.1), extracellular matrix (P < 0.02, n = 9, fold change -1.5 to -3.9), cell adhesion (P < 0.05, n = 10, fold change -1.5 to -2.7), and calcium ion binding (P < 0.01, n = 13, fold change -1.5 to -3.0). Array findings were verified by quantitative PCR for 36 genes. These data combined with previous findings strengthen the evidence for diabetes-induced cardiac gene expression changes involved in cell growth and development, oxidoreductase activity, and the extracellular matrix and also point out other gene groups not previously identified as being affected, such as those involved in calcium ion homeostasis.

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.

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.

Gene expression microarrays and respiratory muscles.

The routine measurement of the expression of tens of thousands of gene transcripts, simultaneously, is a defining advance of the last decade which has been made possible by microarray technology. Using this very powerful approach, a pattern has emerged from a number of studies that suggest a molecular niche for the diaphragm which is quite different from that occupied by limb muscle. All indications are that this is true not only in regard to differential gene transcription patterns in healthy muscles but also in the changes in transcription occurring in association with different diseases. Furthermore, respiratory muscle mounts a rich gene expression response to a number of disturbances, be they primary genetic defects (e.g. various types of muscular dystrophies) or non-genetic perturbations (e.g. controlled mechanical ventilation). Large numbers of genes undergo altered levels of transcription, ranging from tens to hundreds (typical) to thousands. These genes are involved in diverse cellular processes, such as contraction, intermediate metabolism, oxidative stress, apoptosis and cellular adhesion. Functional groups of genes identified as having changed expression differ in many respects from one disease to another. Previously identified pathways of muscle injury and repair are often perturbed to greater extents than previously anticipated, and processes not previously suspected of having important roles in the pathophysiology of specific disorders have been identified. Elucidation of these under-appreciated molecular events may lead to novel therapeutic interventions based on disrupting the downstream adverse consequences of the primary event or facilitating events which ameliorate the injury and/or promote muscle healing.