Michelle Moyer

Changes in pharyngeal respiratory muscle force produced by K+ channel blockade

The purpose of the present study was to determine whether the contractility of pharyngeal respiratory muscles can be augmented by altering membranous K+ channel conductance. The effects on twitch force of two K+ channel blockers, tetraethylammonium (TEA, 10 mM) and 4-aminopyridine (4-AP, 0.3 mM), were examined in vitro for sternohyoid and diaphragm muscle strips. Both agents augmented isometric twitch force of both muscles. In response to TEA twitch force of the sternohyoid muscle increased significantly more than that of the diaphragm (by 33 +/- 7 vs. 9 +/- 1%, P = 0.004), whereas with 4-AP the increase in twitch force of the sternohyoid muscle was comparable to that of the diaphragm (55 +/- 15 vs. 64 +/- 6%, P = 0.50). 4-AP shifted the force-frequency relationship of both muscles leftward but did not alter peak tetanic force, so that force with 4-AP exceeded that without drug at stimulation frequencies below 60 Hz. In contrast TEA reduced force at stimulation frequencies > 20 Hz. The isometric contraction times of both muscles was variably prolonged, more so with 4-AP (by 30 +/- 15% for the sternohyoid and 32 +/- 3% for the diaphragm) than with TEA (by 9 +/- 2% for the sternohyoid and 5 +/- 2% for the diaphragm). For the group of muscles and K+ channel blockers, the degree of augmentation of twitch force correlated with the degree of prolongation of contraction time (r = 0.82, P < 0.001), consistent with blocking delayed rectifier K+ channels as the mechanism of increasing muscle force.

Modulation of diaphragm action potentials by K+ channel blockers

K(+) channels regulate diaphragm contractility. The present study examined the electrophysiological mechanisms accounting for diversity among K(+) channel blockers in their inotropic actions on the diaphragm. Rat diaphragmatic muscle fibers were recorded intracellularly in vitro at 37 degrees C. Apamin and charybdotoxin (Ca2+)-activated K(+) channel blockers) did not alter resting membrane potential or action potentials. Glibenclamide (ATP-sensitive K(+) channel blocker) slowed action potential repolarization by 12% (P<0.05) and increased action potential area by 25% (P<0.005). Tetraethylammonium (which blocks several types of K(+) channels) increased action potential overshoot by 20% (P<0.01) and prolonged action potential rise time by 17% (P<0.02). 4-Aminopyridine and 3,4-diaminopyridine (which also block several types of K(+) channels) slowed action potential repolarization by 163% (P<0.0001) and 253% (P<0.0001), and increased action potential area by 183% (P<0.0001) and 298% (P<0.0001), respectively. Slowing of repolarization for the aminopyridines was especially marked at voltages approaching resting membrane potential, thereby changing action potential repolarization from a first to a second order decay. Previously reported variability in inotropic effects among K(+) channel blockers correlated significantly with the extent to which they slowed action potential repolarization and increased action potential area, but not with changes in other action potential properties.

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.

Streptozotocin-diabetes alters action potentials in rat diaphragm.

This study tested the hypothesis that diabetes alters diaphragm action potentials and electrophysiological responses to K(+) channel blockade. Intracellular recordings were performed in vitro in diaphragm fibers from streptozotocin-induced diabetic and normal Wistar rats (glucose 670+/-31 vs. 252+/-14 mg/dl). Comparing diabetic to normal muscle properties, resting membrane potential was significantly depolarized (-72.2+/-0.8 vs. -77.4+/-1.1 mV), action potential 50% repolarization time was significantly accelerated (0.33+/-0.01 vs. 0.39 +/-0.01 msec), and action potential area was significantly decreased (59.4+/-2.3 vs. 70.7+/-2.2 mV msec). The K(+) channel blocker 3,4-diaminopyridine (DAP) depolarized resting membrane potential of normal but not diabetic muscle. DAP significantly prolonged action potential repolarization and significantly increased action potential area, but significantly more in normal than diabetic muscle. These data indicate that diabetes shortens diaphragm action potentials, which appears to be due to altered K(+) channels.

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.

Altered diaphragm muscle action potentials in Zucker diabetic fatty (ZDF) rats.

The Zucker diabetic fatty (ZDF) rat is a model of type 2 diabetes, being characterized by obesity, diabetes, and dyslipidemia. In vitro studies tested the hypothesis that diaphragm muscle from ZDF rats has abnormal resting membrane potential and action potentials, similar to type 1 diabetic rodents. Resting membrane potential was comparable for muscle from ZDF and control rats. Diaphragm from ZDF rats had augmented action potential peak height (92.1 mV versus 82.4 mV, P<0.00001), overshoot (15.6 mV versus 8.1 mV, P<0.001) and area (80.7 mV ms versus 68.6 mV ms, P<0.001) compared with that from controls. Action potential rate of depolarization and repolarization were not affected. The K(+) blocker, 3,4-diaminopyridine, augmented action potential duration and area of muscle from ZDF and controls, but without significant differences between animal groups. These findings in ZDF rats contrast with type 1 diabetic rats, suggesting that isolated hyperglycemia differs from hyperglycemia combined with other metabolic perturbations with respect to diaphragm electrophysiological derangements.

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.

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‐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 Hills 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.

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.

Sternohyoid muscle fatigue properties of dy/dy dystrophic mice, an animal model of merosin-deficient congenital muscular dystrophy.

Humans with merosin-deficient congenital muscular dystrophy have both sucking problems during infancy and sleep-disordered breathing during childhood. We hypothesized that merosin-deficient pharyngeal muscles fatigue faster than normal muscles. This was tested in vitro using sternohyoid muscle from an animal model of this disease, the dy/dy dystrophic mouse. Isometric twitch contraction and half-relaxation times were similar for dy/dy and normal sternohyoid. However, rate of force loss during repetitive 25-Hz train stimulation was markedly diminished in dystrophic compared with normal sternohyoid muscle. Furthermore, force potentiation, which occurred during the early portion of the fatigue-inducing stimulation, had a longer duration in dystrophic compared with normal muscle (approximately 60 versus 20 s). As a result of these two processes, at the end of 2 min of stimulation, force of dystrophic muscle had decreased by 8 ± 5% and that of normal muscle by 69 ± 4% (p < 0.0001). The potassium-channel blocker, 3,4-diaminopyridine, increased force of dy/dy sternohyoid muscle during twitch and 25-Hz contractions by 148 ± 20% (p < 0.00001) and 109 ± 18% (p < 0.00002), respectively. During repetitive 25-Hz stimulation, force of 3,4-diaminopyridine-treated dystrophic muscle remained significantly higher than that of untreated muscle, despite the early force potentiation being eliminated and fatigue being accelerated. Thus, merosin deficiency reduces fatigue and prolongs the duration of force potentiation. The latter alterations may partially preserve the integrity of upper airway muscle function, without which the severity of pharyngeal complications (feeding problems, sleep-related respiratory dysfunction) might be even more pronounced in the human merosin-deficient congenital muscular dystrophies.