Erik van Lunteren

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.

Comparative effects of aging on pharyngeal and diaphragm muscles

We hypothesized that aging is associated with alterations in pharyngeal muscle structural and contractile properties. Sternohyoid and geniohyoid muscles from young (3-4 months) and old (20-21 months) Fischer 344 rats were compared with diaphragm muscle. The pharyngeal muscles had significantly lower proportions of slow oxidative (SO) fibers compared to the diaphragm, and the percentage of fast glycolytic (FG) fibers was significantly higher in the sternohyoid than in both the geniohyoid and the diaphragm. With senescence, there was a small but significant increase in the proportion of FG fibers and a corresponding reduction in the proportion of fast oxidative glycolytic (FOG) fibers in all three muscles. The sternohyoid muscle had significantly faster isometric contractile kinetics and lower fatigue indexes than the diaphragm. Aging was associated with significant worsening of sternohyoid endurance, but no significant alterations in sternohyoid twitch kinetics or diaphragm properties. These results indicate that in rats the pharyngeal dilator muscles have larger proportions of fast fibers, fast contractile kinetics and worse endurance than the diaphragm. Furthermore, aging was associated with a shift to a higher proportion of FG fibers with a concomitant reduction in proportion of FOG fibers, as well as a decline in pharyngeal muscle endurance.

Action of nicotine on the respiratory activity of the diaphragm and genioglossus muscles and the nerves that innervate them

Nicotine is known to alter respiration by stimulating peripheral chemoreceptors and receptors within the brain. In this study the sites of action and the effects of nicotine on hypoglossal nerve activity were compared to its effects on phrenic activity in paralyzed, vagotomized and chloralose-anesthetized cats. Since anesthesia is known to affect respiratory responses, we also compared the effects of intravenous nicotine given to conscious unsedated cats on genioglossus and diaphragm electrical activity. In eight conscious animals intravenous doses of nicotine ranging between 10 ng and 200 micrograms increased genioglossus activity significantly more than diaphragm activity. Studies in 26 anesthetized animals included injection of nicotine, intravenously, in the lateral ventricles, and application of nicotine to the ventrolateral surface of the medulla (the putative site of the central chemoreceptors) before and after section of the carotid sinus nerves. With all these interventions, changes in hypoglossal nerve activity were significantly greater than changes in phrenic nerve activity. The responses to nicotine could be blocked by application of hexamethonium to the ventrolateral medullary surface or by cooling the same area. The results indicate that: nicotine increases hypoglossal nerve activity by both its peripheral and central effects; nicotine has differential effects on different respiratory muscles and nerves; and the central action of nicotine may be mediated largely through receptors located near the ventral medullary surface.

Activity of upper airway muscles during augmented breaths

The effect of augmented breaths on the electrical activity of upper airway (UAW) muscles was studied in fourteen spontaneously breathing anesthetized dogs. Moving average traces of the electrical activity recorded from the genioglossus (GG), the posterior cricoarytenoid (PCA), and the alar portion of the nasalis muscle (AN) were compared to tracings of diaphragm electrical activity. During augmented breaths the electrical activity of the diaphragm showed the characteristic biphasic pattern previously described: an initial phase following the contour of a normal breath (phase I) and an augmented phase arising near the crest of the initial phase (phase II). During all augmented breaths, the GG, PCA and AN showed the same biphasic pattern as the diaphragm. The normally rounded shape of UAW muscle EMG activity during control breaths changed to a more sharply peaked form during the second phase of the augmented breath. Onset of activity of all UAW muscles studied preceded that of the diaphragm; during control breaths, the average interval was 0.29 sec for the PCA, 0.25 sec for the GG and 0.14 sec for the AN (P less than 0.05). The amount of pre-activation was decreased to less than 0.10 sec during the second phase of the augmented breath. The slopes and amplitudes of phase I were similar to that of control breaths. The peak EMG activity of the augmented breath was 214% of the control breaths for the diaphragm, 247% for the GG, 168% for the AN and 161% for the PCA (P less than 0.005 for GG, P less than 0.001 for the others). During hyperoxic hypercapnia the slopes and amplitudes of phase II remained nearly constant for all four muscles, whereas the slopes and amplitudes of phase I changed with the chemical drive just as in control breaths. UAW resistance, recorded in five additional spontaneously breathing anesthetized dogs, was 32% less during inspiration than expiration during control breaths, and 31% less during phase I of augmented breaths; there was a further 18% decrease during phase II of augmented breaths (P less than 0.001). The results suggest that mechanisms responsible for augmented breaths act similarly on upper airway muscles and the diaphragm.

Comparison of the response of diaphragm and upper airway dilating muscle activity in sleeping cats

The steady state and transient effects of hyperoxic hypercapnia on the electromyographic activities of the genioglossus (GG), posterior cricoarytenoid (PCA), and diaphragm (D) were studied in cats instrumented with chronically implanted electrodes during non-rapid eye movement sleep. Hypercapnia (inhalation of 3.4 and 7.4% CO2 in O2) increased the phasic electrical activity occurring during inspiration of all three muscles. Activities of the PCA and D increased in a parallel fashion with increasing CO2. The GG responded to steady state CO2 inhalation alinearly, with larger increases in activity occurring when CO2 was raised from 3.4 to 7.4% than when CO2 was increased from 0 to 3.4%. When gas mixtures containing CO2 were given, the GG reached its new steady state level more slowly than the D or PCA, and when CO2 was rapidly removed from the inspired gas mixture, the GG attained its steady state sooner than the D. These results suggest that in sleeping cats, hypercapnia does not affect either transient or steady state responses of the GG in the same way as the D or the PCA. These differences seem to be explained largely by different threshold characteristics of hypoglossal and phrenic neurons.

Responses of hypoglossal and phrenic nerves to decreased respiratory drive in cats

Agents which depress respiration, such as alcohol, seem to increase the occurrence of obstructive apneas during sleep. It has been proposed that upper airway obstruction can result from an imbalance in the activity (or forces) produced by the upper airway muscles versus the chest wall muscles so that upper airway passages might be blocked when a disproportionate decrease in upper airway muscle activity occurs. This study examines the hypothesis that depression of respiration affects the activity of the hypoglossal nerve (the motor nerve to the tongue) more than the activity of the phrenic nerve (the motor nerve to the diaphragm). In addition, we examined the role of the putative central chemoreceptor area on the ventrolateral medullary surface (VMS) in maintaining phrenic and hypoglossal discharge. In chloralose-anesthetized, artificially ventilated, paralyzed cats, three methods of reducing respiratory drive were studied: hyperoxic hypocapnia (produced by mechanical hyperventilation), the application to the intermediate area of the ventral medullary surface of the respiratory depressant GABA and its agonist muscimol, and cooling the same area of the VMS (using a water-cooled thermode). All these interventions decreased hypoglossal nerve activity more than phrenic nerve activity (range of p values: p less than 0.001 to p less than 0.01). Moreover, the reduction in hypoglossal activity was greater with GABA and muscimol than with the other two maneuvers; this was statistically significant for both GABA versus VMS cooling (p less than 0.02) and muscimol versus VMS cooling (p less than 0.01). These results show that respiratory depression can differentially affect hypoglossal and phrenic nerve activity.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Heterogeneity within geniohyoid motor unit subpopulations in firing patterns during breathing

Respiratory motor units (MU) segregate into subpopulations, which differ in firing patterns during resting and stimulated breathing. For phrenic/diaphragm MUs, diversity also exists within subpopulations, and is greater for late than early-onset MUs. The present study characterized the extent of diversity within upper airway respiratory MU subpopulations by recording geniohyoid MUs in anesthetized cats. Inspiratory MUs (I-MU, n=21) had a wide range of firing durations (coefficient of variation (CV)=42%). In contrast, inspiratory-expiratory MUs (I/E-MU, n=19) had a narrow range of firing durations during inspiration (CV=13%), but a wide range of firing durations during expiration (CV=36%). Mean firing frequency had similar degrees of diversity among units for I-MU and I/E-MU (CV=31-40%). For I-MU firing duration correlated with mean firing frequency, whereas no such relationship was apparent for I/E-MU. Single-breath end-expiratory airway occlusion decreased heterogeneity in firing duration during inspiration and increased it during expiration, whereas end-inspiratory airway occlusion decreased heterogeneity during expiration. In conclusion, (a) there is considerable diversity within geniohyoid MU subpopulations receiving respiratory drive; (b) the degree of diversity within subpopulations differs for I-MU and I/E-MU; and (c) diversity within subpopulations in timing of activity is modulated by single-breath airway occlusion.

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.