The Effects of Experimental Sleep Apnea on Cardiac and Respiratory Functions in 6 and 18 Month Old Dystrophic (mdx) Mice.

Duchenne muscular dystrophy (DMD) is a fatal disease where over 90% of patients succumb to respiratory or cardiac failure. Sleep apnea and sleep disordered breathing (SDB) are noted in a plurality of DMD patients, and the resulting nocturnal episodic hypoxia (EH) cannot be ruled out as a contributing factor to cardiac and respiratory dysfunction. In this study, we investigated the impact of long-term episodic hypoxia, which mimics the cyclic hypoxia seen in sleep apnea, on cardiac and respiratory function in a murine model of DMD (mdx mice). Since the severity and prevalence of sleep apnea in DMD increases with age, we studied the impact of EH on young (6-month) and on older (18-month) mdx mice. Mice were either exposed for 12 weeks to EH (8 hours/day, 5 days/week) or to room air. We noted a significant increase in left ventricular (LV) dilatation (transthoracic echocardiography) on EH exposure in both age groups, but reduced LV contractility was seen only in 6-month old mice. With EH exposure, an increased fibrosis (hydroxyproline) was noted in both cardiac and diaphragm muscle in 18-month but not 6-month old mice. No significant change in relative diaphragm strength (in-vitro) was noted on EH exposure in 18-month old mice. In contrast, EH exposed 6-month old mice showed a significant increase in relative diaphragm strength. EH exposure did not result in any significant change in ventilatory parameters (barometric plethysmography) in awake 6-month old mdx mice. In contrast, 18-month old mdx mice showed considerable ventilatory dysfunction, consistent with reduced ventilatory reserve. Our findings highlight that sleep apnea impacts respiratory and cardiac function in muscular dystrophy, and that EH can have divergent effects on both systems. To our knowledge, this is the first comprehensive study to investigate the impact of EH on cardiac and respiratory function in mdx mice.

Exercise capacity in hamsters with elastase-induced emphysema compared to normal controls.

The purpose of this study was to determine whether hamsters with elastase-induced emphysema (EMP) would demonstrate a reduction in exercise capacity compared to control (CON) hamsters and whether changes in activity levels, muscle function and structure could explain any changes in exercise capacity. Peak oxygen consumption and daily activity levels were measured on two occasions. Inspiratory capacity under deep anesthesia, in vitro measurements of muscle force and fatigability for the diaphragm (DIA) and extensor digitorum longus (EDL) and fiber proportions, muscle cross-sectional area and fiber specific SDH activity from the DIA, EDL and vastus lateralis (VLA) were obtained. Inspiratory capacity was 60% higher in the EMP compared to CON hamsters (p=0.0004). Activity levels and exercise capacity were not significantly different between EMP and CON hamsters. Muscle strength and fatigability, fiber proportions, muscle cross-sectional area and fiber specific SDH activity were similar between EMP and CON hamsters. In conclusion, in hamsters, elastase-induced emphysema did not reduce maximal exercise capacity.

In-situ mechanical characteristics of the tongue are not altered in the obese Zucker rat.

STUDY OBJECTIVES: Obese Zucker rats have more collapsible isolated upper airways, compared with their lean counterparts. The functional characteristics of the tongue as a potential mechanism for the enhanced upper airway collapsibility in the obese Zucker rat are unknown. This study measured the functional characteristics of the tongue muscle in lean and obese Zucker rats. DESIGN: In-situ tongue force (twitch and peak) and fatigability were measured in anesthetized obese and lean Zucker rats. SETTING: Animal housing facility at the University of Buffalo. SUBJECTS: Eight lean and eight obese Zucker rats. INTERVENTION: Tongue force and fatigability were measured before, during, and following cocontraction of the tongue protrudor and retractor muscles via direct stimulation of the common hypoglossal nerve. MEASUREMENTS AND RESULTS: Obese rats were significantly heavier than their lean counterparts (718 +/- 101 gm vs. 545 +/- 32, P < 0.05). Total force production at all stimulation frequencies was not different between lean and obese Zucker rats before or after fatigue (P = 0.436). Forces were significantly reduced at the end of the 5-minute stimulation period (P < 0.001) and returned to baseline within 1 minute after fatigue in both lean and obese rats. At the end of the fatigue protocol, tongue force averaged 63.3% +/- 13.8% and 72.3% +/- 17.8% of the initial force in obese and lean rats respectively (P= 0.85). CONCLUSION: Obesity does not alter the in-situ force production of the tongue muscle. Thus, increases in collapsibility of the isolated upper airway previously noted in obese Zucker rats cannot be ascribed to upper airway muscle dysfunction or enhanced fatigability.

Aging increases upper airway collapsibility in Fischer 344 rats.

The upper airway muscles play an important role in maintaining upper airway collapsibility, and the incidence of sleep-disordered breathing increases with age. We hypothesize that the increase in airway collapsibility with increasing age can be linked to changes in upper airway muscle mechanics and structure. Eight young (Y: 6 mo) and eight old (O: 30 mo) Fischer 344 rats were anesthetized and mechanically ventilated, and the pharyngeal pressure associated with flow limitation (Pcrit) was measured 1) with the hypoglossal (cnXII) nerve intact, 2) following bilateral cnXII denervation, and 3) during cnXII stimulation. With the cnXII intact, the upper airways of older rats were more collapsible compared with their younger counterparts [Pcrit = -7.1 +/- 0.6 (SE) vs. -9.5 +/- 0.7 cmH2O, respectively; P = 0.033]. CnXII denervation resulted in an increase in Pcrit such that Pcrit became similar in both groups (O: -4.2 +/- 0.5 cmH2O; Y: -5.4 +/- 0.5 cmH2O). In all rats, cnXII stimulation decreased Pcrit (less collapsible) in both groups (O: -11.3 +/- 1.0 cmH2O; Y: -10.2 +/- 1.0 cmH2O). The myosin heavy chain composition of the genioglossus muscle demonstrated a decrease in the percentage of the IIb isoform (38.3 +/- 2.5 vs. 21.7 +/- 1.7%; P < 0.001); in contrast, the sternohyoid muscle demonstrated an increase in the percentage of the IIb isoform (72.2 +/- 2.5 vs. 58.4 +/- 2.3%; P = 0.001) with age. We conclude that the upper airway becomes more collapsible with age and that the increase in upper airway collapsibility with age is likely related to altered neural control rather than to primary alterations in upper airway muscle structure and function.

Episodic hypoxia exacerbates respiratory muscle dysfunction in DMD(mdx) mice.

Many patients with Duchenne muscular dystrophy (DMD) are eventually diagnosed with sleep-disordered breathing (SDB). SDB is associated with reduced ventilation, decreased arterial oxygen tension, and increased respiratory muscle recruitment during sleep, factors that could be especially detrimental to respiratory muscles in DMD. To assess whether SDB impacts dystrophin-deficient respiratory muscle function and fibrosis, diaphragm strength, and collagen content were evaluated in dystrophic mice (Dmd(mdx)) exposed to experimental SDB. Diurnal exposure to episodic hypoxia resulted in a 30% reduction in diaphragm strength without affecting collagen content. Episodic hypoxia secondary to SDB can exacerbate respiratory muscle dysfunction in DMD.

Intermittent hypoxia reduces upper airway stability in lean but not obese Zucker rats.

Obstructive sleep apnea involves intermittent periods of airway occlusions that lead to repetitive oxygen desaturations. Exposure to chronic intermittent hypoxia (IH) in rats increases diurnal blood pressure and alters skeletal muscle physiology. The impact of IH on upper airway muscle function is unknown. We hypothesize that IH exposure increases upper airway collapsibility in rats due to alterations of the muscles surrounding the upper airway. Lean and obese rats were exposed to cyclic alterations in O(2) levels (20.6%-5%) every 90 s, 8 h/day for 6 days/wk for 12 wk. Following the exposure period, arterial pressure was recorded via the tail artery in conscious unrestrained rats. Mean arterial pressure was increased in lean IH but not in obese IH-exposed Zucker rats (P < 0.05). The pharyngeal pressure associated with airway collapse (P(crit)) was measured under anesthesia during baseline conditions and then during supramaximal stimulation of the hypoglossal nerve (cnXII). Baseline P(crit) was more positive (more collapsible) in lean but not obese rats following 12 wk of IH (P < 0.05), while supramaximal stimulation of cnXII increased airway stability (decreased P(crit)) in both lean and obese Zucker rats following IH to levels that were similar to their respective room air controls. The in vitro peak tension and the expression of the individual myosin heavy chain isoforms from the upper airway muscles were unaltered following IH. We conclude that IH leads to increases in baseline collapsibility in lean Zucker rats exposed to IH by nonmyogenic mechanisms.

A comparison of factors associated with collagen metabolism in different skeletal muscles from dystrophic (mdx) mice: impact of pirfenidone.

Skeletal muscles in mdx mice exhibit differential degrees of pathological changes and fibrosis. The purpose of this study was to examine differences in various indices of collagen metabolism in skeletal muscles with widely different functions and activity profiles in mdx mice, and to determine whether pirfenidone would attenuate the development of fibrosis. Mice in the pirfenidone group were orally fed pirfenidone (500 mg/kg) daily for 4 weeks. Marked differences were noted in hydroxyproline concentration between muscles, which could not be explained solely by the level of type I collagen and transforming growth factor-beta1 (TGF-beta1) mRNA. In normal mice, matrix metalloproteinase (MMP)-2 mRNA was significantly higher in the gastrocnemius than in the diaphragm or genioglossus muscles, suggesting that collagen degradation plays an important role in regulating collagen accretion in skeletal muscle. In mdx mice, the levels of both MMP-2 and MMP-9 mRNA were significantly elevated relative to control, although the response was muscle specific. Pirfenidone treatment resulted in a significant reduction in the level of hydroxyproline concentration across all muscles, although the effect was small. Results from this study reveal intrinsic dissimilarities in collagen metabolism between functionally different skeletal muscles. Moreover, the pharmacological use of pirfenidone may be beneficial in preventing fibrosis in muscular dystrophy.

Adenosinergic modulation of ventilation in obese zucker rats.

OBJECTIVES: The goal of our study was to determine whether altered adenosinergic mechanisms contribute to the depressed ventilatory response observed in obese Zucker rats. RESEARCH METHODS AND PROCEDURES: Eight lean and eight obese Zucker rats were studied at 7 to 8 weeks of age. Ventilation (V(E)) during room air, during 5-minute hypercapnic (7% CO(2), balance O(2)), and during 30-minute sustained hypoxic (10% O(2)) exposures were sequentially measured by the barometric method on three separate occasions after the randomized blinded administration of equal volumes of either saline (control), 8-(p-sulfophenyl)-theophylline (8-PST, 7 mg/kg, peripheral adenosine antagonist), or aminophylline (AMPH, 15 mg/kg, peripheral and central adenosine antagonist). RESULTS: During room air and hypercapnic exposures, AMPH (but not 8-PST) significantly (p < 0.05) increased V(E) in both lean and obese rats. During acute (2 minute) hypoxic exposure, 8-PST (but not AMPH) significantly depressed V(E) in lean rats. In contrast, AMPH (but not 8-PST) selectively increased V(E) in obese rats. During sustained (10 to 30 minutes) hypoxic exposure, neither AMPH nor 8-PST administration altered V(E) in lean rats. In contrast, AMPH (but not 8-PST) selectively increased V(E) during the late response in obese rats. DISCUSSION: Our findings indicate that obese rats possess altered adenosinergic modulation of ventilatory responses to acute and sustained hypoxia in two opposite ways. We conclude that the reduced hypoxic ventilatory response observed in obese Zucker rats is attributed to depressed adenosinergic peripheral excitatory mechanisms and to enhanced adenosinergic central depression mechanisms, both of which contribute to the blunted ventilatory response in obesity.

Systemic administration of serotonin 2A/2C agonist improves upper airway stability in Zucker rats.

The effects of [+/-]-2,5-dimethoxy-4-iodoaminophentamine, a serotonin(2A/2C) receptor agonist, on pharyngeal airflow mechanics were examined in isoflurane-anesthetized lean and obese Zucker rats. The pharyngeal pressure associated with flow limitation, maximum inspiratory flow, oronasal resistance, genioglossus muscle activity, and arterial blood pressure (BP) were measured before and after the intravenous administration of the agonist. A robust activation of the genioglossus muscle in all lean and obese rats was associated with decreased upper airway (UA) collapsibility (p < 0.05), unchanged maximum flow, and increased oronasal resistance (p < 0.05) in both groups. The changes in UA mechanics and BP after the drug were similar in lean and obese rats. The serotonin agonist had no effect on UA mechanics in a group of paralyzed (pancuronium bromide) rats, despite similar elevations in BP. There was a smaller decrease (p < 0.05) in UA collapsibility that was also associated with increased upstream resistance when the drug was administered after bilateral hypoglossal nerve transection. We conclude that systemic administration of a serotonin(2A/2C) receptor agonist improves UA collapsibility predominantly, but not exclusively, via stimulation of the hypoglossal nerves and also increases upstream resistance, at least in part, through activation of nonhypoglossal motoneuronal pools innervating the UA muscles.

Ventilatory dysfunction in mdx mice: impact of tumor necrosis factor-alpha deletion.

Muscular dystrophy is associated with inflammation and fiber necrosis in the diaphragm that may alter ventilatory function. The purpose of this study was to determine to what extent in vivo ventilatory function in dystrophic (mdx) mice was compromised and to assess the impact of deletion of tumor necrosis factor-alpha (TNF-alpha), a known proinflammatory cytokine, on ventilatory function, diaphragm contractility, and myosin heavy chain (MHC) distribution in 10-12-month-old mdx mice. Although the resting ventilatory pattern did not significantly differ between control and mdx mice, the ventilatory response to hypercapnia in mdx mice was significantly attenuated. Elimination of TNF-alpha significantly improved the hypercapnic ventilatory response and diaphragm muscle maximal isometric force. Long-term TNF-alpha deletion also altered the myosin heavy chain isoform profile of the diaphragm. These data indicate that a blunted ventilatory response to hypercapnia exists in mdx mice, and that TNF-alpha influences the progressive deterioration of diaphragm muscle in mdx mice.

Adaptation of upper airway muscles to chronic endurance exercise.

We tested the hypothesis that chronic endurance exercise is associated with the recruitment of four major upper airway muscles (genioglossus, digastric, sternohyoid, and omohyoid) and results in an increased oxidative capacity and a fast-toward-slow shift in myosin heavy chain (MHC) isoforms of these muscles. Female Sprague-Dawley rats (n = 8; 60 days old) performed treadmill exercises for 12 weeks (4 days/week; 90 minutes/day). Age-matched sedentary female rats (n = 10) served as control animals. Training was associated with an increase (p < 0.05) in the activities of both citrate synthase and superoxide dismutase in the digastric and sternohyoid muscles, as well as in the costal diaphragm. Compared with the control animals, Type I MHC content increased (p < 0.05) and Type IIb MHC content decreased (p < 0.05) in the digastric, sternohyoid, and diaphragm muscles of exercised animals. Training did not alter (p > 0.05) MHC phenotype, oxidative capacity, or antioxidant enzyme activity in the omohyoid or genioglossus muscle. These data indicate that endurance exercise training is associated with a fast-to-slow shift in MHC phenotype together with an increase in both oxidative and antioxidant capacity in selected upper airway muscles. It seems possible that this exercise-mediated adaptation is related to the recruitment of these muscles as stabilizers of the upper airway.

Diaphragm plasticity following intrinsic laryngeal muscle denervation in rats.

PURPOSE: During inspiration, recruitment of the intrinsic laryngeal muscles (ILM) reduces the inspiratory load on the ventilatory pump muscles. The purpose of our study was to determine 1) whether the diaphragm adapts to denervation of the ILM, and 2) whether the additional stimulus of exercise training affects the degree to which the diaphragm adapts to ILM denervation. METHODS: Thirty-six male Sprague-Dawley rats (2 months) were randomly divided into sedentary control (SC), sedentary-denervated (SD), and exercise-denervated (ED) groups. Control animals underwent sham operations, whereas ILM-denervated animals underwent bilateral transection of the recurrent laryngeal nerves. Three weeks after surgery, animals in the ED group performed a treadmill training protocol for a period of 6 wk. RESULTS: Denervation (SD and ED animals) of the ILM significantly increased diaphragm citrate synthase activity (20%), in vitro endurance, and time to peak twitch tension (15%), and reduced (13%) peak tetanic tension (Po, N x cm(-2)). No independent training effect over and above the effects attributed to denervation of ILM were noted in ED animals. CONCLUSION: The results highlight the role of vocal cord dilator function during both eupnea and exercise-induced hyperpnea.

Dopaminergic modulation of ventilation in obese Zucker rats.

To investigate the hypothesis that the impaired respiratory drive noted in morbid obesity was attributable to altered dopaminergic mechanisms acting on peripheral and/or central chemoreflex sensitivity, seven obese and seven lean Zucker rats were studied at 11 wk of age. Ventilation (VE) was measured by the barometric technique during hyperoxic (100% O(2)), normoxic (21% O(2)), hypoxic (10% O(2)), and hypercapnic (7% CO(2)) exposures after the administration of vehicle (control), haloperidol [Hal, 1 mg/kg, a central and peripheral dopamine (Da) receptor antagonist], or domperidone (Dom, 0.5 mg/kg, a peripheral Da receptor antagonist). In both lean and obese rats, Hal increased tidal volume and decreased respiratory frequency during hyperoxia or normoxia, resulting in an unchanged VE. In contrast, Dom did not affect tidal volume, frequency, or VE during hyperoxia or normoxia. During hypoxia, however, VE significantly increased from 1,132 +/- 136 to 1,348 +/- 98 ml. kg(-1). min(-1) (P < 0.01) after the administration of Dom in obese rats, whereas no change was observed in lean rats. Hal significantly decreased VE during hypoxia compared with control in lean but not obese rats. In both lean and obese rats, Hal decreased VE in response to hypercapnia, whereas Dom had no effect. Our major findings suggest that peripheral chemosensitivity to hypoxia in obese Zucker rats is reduced as a result of an increased dopaminergic receptor modulation in the carotid body.