Abstract

Upper airway muscles regulate upper airway patency. Obstructive sleep apnea is caused by upper airway collapse, and its incidence increases with age and is higher in men than women. The reasons for this are unknown, as little is known about the effects of age and gender on upper airway muscle. Isometric contractile properties were determined using strips of geniohyoid and sternohyoid muscles from young and old, male and female rats in physiological saline solution at 30°C. There were no differences between the male and female rats in any of the contractile properties of either muscle, and this was true for both young and old animals. Aging had no effect on sternohyoid contractile properties, but geniohyoid force was greater in old than in young rats.

Decision Editor: Jay Roberts, PhD

GENDER differences in skeletal muscle cross-sectional area and muscle fiber type and number have been shown to occur in various species including humans, rats, and mice (1)(2)(3)(4)(5). However, evidence for effects of gender on muscle contractile properties has been equivocal. A number of studies have shown that men develop larger specific force compared to women (6)(7), but others have found that there is no difference (3)(8)(9). This relationship is also complicated by the effect of aging. Thus, there is evidence, for example, that aging reduces limb muscle force in men (10) but not in women (11). The muscles of the upper airway (UA) play an important role in maintaining the patency of the UA (12). Obstructive sleep apnea (OSA) is a major clinical disorder (13)(14) in which there is episodic apnea during sleep because of the collapse of the UA. This collapse is due to the subatmospheric pressure generated in the airway during inspiration as a result of diaphragmatic contraction and the fact that the anterior portion of the pharynx is not supported by rigid structures (12). Normally, contraction of the UA dilator muscles such as the genioglossus, geniohyoid, and sternohyoid muscles counteracts this collapse by stabilizing and dilating the airway (12). The incidence of OSA in men is two- to tenfold greater than in women (14), and the incidence shows a positive relationship with age (13). The reasons for these gender and age effects are unknown. It is known that there are gender differences in human UA muscle activity (15), but the effects of aging on respiratory control are controversial (16)(17)(18)(19)(20). One possible explanation for the gender and age differences in the incidence of OSA is that there are gender and age differences in UA muscle contractile properties. However, the effects of age and/or gender on human UA muscle function are unknown. Van Lunteren, Vafaie, and Salomone (21) have shown that aging reduces sternohyoid but not diaphragm endurance in rats. We have previously shown that there are no gender differences in UA muscle contractile properties in young rats (22), but it is not known if gender influences UA muscle contractile properties in old animals. The present investigation examines the in vitro contractile properties of the geniohyoid and sternohyoid muscles in young and old, male and female rats. The only animal model of OSA in humans is the English bulldog (23). Rats are known to have central sleep apnea (24), but it is not known if they have OSA. However, because of the time necessary for longitudinal studies in humans and indeed in the dog model of OSA, the rat is a useful model of age-related effects on UA muscles, especially because it has been shown that age-related changes in skeletal muscle function in rats have a number of similarities to humans (25).

Methods

Experiments were carried out on 44 (21 male and 23 female), 10–14-week-old and 40 (20 male and 20 female), 19–20-month-old Wistar rats. Animals were anesthetized (40 mg·kg−1), tracheostomized, and artificially ventilated. A femoral artery and vein were cannulated with the aid of a microscope in order to record arterial blood pressure and to administer supplementary anesthetic, respectively. Core temperature was maintained at 37°C using a thermostatically controlled heating blanket and radiant heat.

With the aid of a microscope, the digastric muscles were separated to reveal the omohyoid muscle; this was cut to expose the underlying geniohyoid muscles, which run as two parallel strips from the midpoint of the mandible to the hyoid bone. The sternohyoid muscles running from the sternum to the hyoid bone were also exposed. The muscles were removed rapidly, and longitudinal strips of 1–2 mm in diameter were suspended vertically in a water-jacketed bath in warmed (30°C), oxygenated (95% O2–5% CO2) physiological saline solution (pH 7.4) containing (in mmol) NaCl 120, KCl 5, Ca gluconate 2.5, MgSO4 1.2, NaH2PO4 1.2, NaHCO3 25, glucose 11.5. One end of the muscle strip was fixed and the other end attached to an isometric force transducer mounted on a micropositioner. Isometric twitch tension, tetanic tension, twitch:tetanic tension ratio, contraction time, half-relaxation time, the tension–frequency relationship, and fatigue were measured using field stimulation (supramaximal voltage, 1 ms duration) with platinum electrodes and recorded using an analogue/digital converter and microcomputer.

Protocol

An equilibration period in the bath of 30 minutes was allowed before any measurements were made. Muscle length was changed in increments of 1 mm using the micropositioner, and optimal length determined (i.e., the length producing maximal twitch tension). The muscle was held for the remainder of the experiment at optimal length. The muscle was then stimulated at 10, 20, 30, 40, 50, 60, 80, and 100 Hz for 300 ms at each frequency, allowing 2 minutes between each stimulus. Ten minutes following this tension-frequency determination, fatigue was induced by stimulation at 30 Hz with 300 ms trains of 0.5 Hz for 5 minutes (26).

Data analysis.

Specific tension was calculated in Newtons per square centimeter of strip cross-sectional area. Cross-sectional area was calculated by weighing the muscle strip after removal from the bath, blotting dry, and dividing this by the product of the optimal length and muscle density, assumed to be 1.06 mg per cubic mm. For the tension–frequency relationship, values were normalized by expressing them at different frequencies as a percentage of the maximal tetanic value. For the fatigue protocol, values were normalized by expressing the force generated by the first pulse of the stimulus train at 1, 2, 3, 4, and 5 minutes as a percentage of the value of the first pulse of the first train. These values, the absolute values for specific twitch and tetanic tension, the twitch:tetanic ratio, and the contraction and half-relaxation times were expressed as mean ± SD. They were used to compare statistically the male and female groups and the young and old groups using two-way (Age × Gender) and three-way (Age × Gender × Time) ANOVA and Fisher's least significant difference test (p < .05 as significant) using a statistics package (Data Desk) which incorporates a correction factor for type I error. An estimate of the sample size by power analysis was based on our previously published value of 0.62 ± 0.25 (mean ± SD) Newtons per square centimeter for geniohyoid twitch tension in young rats (22), a minimum detectable difference of 20% of the mean value and a z value of 1.96 (95% confidence interval). This yielded a value for n of 16.

Results

Geniohyoid.

The twitch and tetanic tension per unit cross-sectional area for the geniohyoid muscle showed no significant change between the male and female groups in either the young or old animals (Table 1 ). However, tetanic tension values were significantly greater in old compared to young rats, true for both males and females. The cross-sectional area, twitch:tetanic tension ratio, contraction time, and half-relaxation time showed no significant differences due to gender or age (Table 1 ). The force–frequency relationship and fatigue curves also showed no significant changes due to age or gender (Fig. 1).

Sternohyoid.

Table 2 shows that neither gender nor age had any significant effect on muscle cross-sectional area, twitch and tetanic tension per unit cross-sectional area, twitch:tetanic tension ratio, contraction time, or half-relaxation time. Similarly, Fig. 2 shows that neither gender nor age had any significant effect on the force–frequency or fatigue curves.

Discussion

The present study confirms our previous findings (22) that there are no gender differences in specific force or in any of the other contractile properties of the geniohyoid or sternohyoid muscles in young rats. These muscles are important dilators of the UA. They cause anterior displacement of the hyoid bone, resulting in dilation and stiffening of the hypopharynx (27). In OSA, the oropharynx is the site of UA collapse in 50% of patients, but the region of collapse also includes the hypopharynx in the remaining 50% (28). Our results also confirm our previous findings that the sternohyoid generates greater force than the geniohyoid (22). This is likely to be because the sternohyoid contains more fast fibers than the geniohyoid (21).

Because aging has been shown to affect muscle contractile properties and because this effect may be influenced by gender (10)(11), we also compared UA muscle contractile characteristics in old, male and female rats. We found that, as in the young animals, gender did not affect UA muscle contractile function. The absence of a gender effect on UA muscle contractile properties in both young and old rats suggests that the cause of the gender difference in the incidence of OSA lies elsewhere. One possible cause would be a gender difference in the structure of the UA, but there is no evidence for such a difference (29)(30). We also found that aging had very little effect on UA muscle contractile properties. Thus, aging was not associated with significant changes in the contractile kinetics of the UA muscles. This is in agreement with the findings of van Lunteren and colleagues (21), who found that sternohyoid contractile kinetics were unaffected by age in rats. The effect of age on the tension–frequency relationships of the geniohyoid and sternohyoid muscles has not been investigated previously. The tension–frequency relationships of the geniohyoid and sternohyoid did not show significant changes with age. The endurance of the geniohyoid and sternohyoid muscles also did not show any significant changes due to age. In contrast, van Lunteren and colleagues (21) found that aging reduced sternohyoid endurance in Fischer 344 rats. This discrepancy may be due to differences in rat strain or in the method used to induce fatigue [40 Hz with 330 ms trains of 1 Hz for 5 minutes (21) compared to 30 Hz with 300 ms trains of 0.5 Hz for 5 minutes in the present experiments].

The effect of age on the contractile force of UA muscles has not been studied previously. We observed that aging increased geniohyoid muscle force production. Significant alterations in the morphology and contractile properties of some skeletal muscles with age have been described previously. In human studies, it has been determined that absolute muscle force decreases with aging (31)(32)(33), and this decrease has been found to correlate to a decrease in muscle mass (32). The decrease in muscle force production is thought to be a result of alterations in fiber recruitment (34), contractile properties (35), or cellular properties such as an age-related change in muscle fiber type composition (33)(35).

Numerous studies have demonstrated a reduction in number and diameter of fast-twitch muscle fibers with age (32)(33)(36), whereas the content and size of the slow-twitch muscle fibers are much less affected, if at all (32)(33)(34)(36). However, loss of muscle force with age is not universally observed (34)(37)(38). Therefore, the effects of aging on muscle force in humans remain unclear.

In rats, there seems to be a preferential age-related loss of fast-twitch, as opposed to slow-twitch, muscle fibers in the limb muscles (39)(40). However, some investigators have reported age-related increases in the number of slow-twitch muscle fibers in rats (39)(41), whereas others have reported no changes in fiber type with age in both fast- and slow-twitch muscles (42), or else no change in slow-twitch fibers but a decrease in fast-twitch fibers (43). Similarly, histological changes in the muscles of rats with aging seem to vary from muscle to muscle. For example, in the rat soleus muscle, slow-twitch fiber percentage decreases with age, whereas the percentage of slow-twitch fibers increases in the extensor digitorum longus (39).

The mechanism for the observed changes in the composition of muscle fibers remains unknown. Larsson (44) has suggested that the muscle fiber transformation may be in response to a change in motoneuron properties related to altered functional demands due to an increase in body weight.

Regarding the effects of age on muscle contractile properties, results in rats, as in humans, have been equivocal. Thus, aging has been shown to either reduce (45), increase (43), or to have no effect (43)(46) on muscle tension development.

There has been only one investigation into the effects of aging on the structural and functional characteristics of UA muscles. This was conducted by van Lunteren and colleagues (21) in rats. The authors looked at the effects of aging on (a) the structural and contractile properties of the diaphragm and sternohyoid muscles, and (b) the structural properties of the geniohyoid. There was a significant increase in the number of the fast glycolytic fibers and a decrease in the number of fast oxidative glycolytic fibers in all three muscles. Similarly, Maltin and colleagues (47) reported a significant increase with aging in the number of fast glycolytic fibers in the diaphragm muscles of rats. With regard to the UA muscles, van Lunteren and colleagues (21) found that the endurance of the sternohyoid muscle worsened with aging, but the contractile kinetics were unaffected. However, the authors did not look at the effect of age on the twitch or tetanic tensions of the geniohyoid or sternohyoid muscles.

In the present experiments, aging had no effect on sternohyoid contractile function, but the tetanic tension per unit cross-sectional area of the geniohyoid muscle showed a significant increase due to age. As mentioned above, the van Lunteren study (21) reported a small but functionally significant increase in the proportion of fast glycolytic muscle fibers in the geniohyoid muscle of old rats; this may underlie the increase in geniohyoid force with age because it has been demonstrated that fast-twitch muscles generate greater tension per unit cross-sectional area than slow-twitch muscle fibers in the rat (48). Furthermore, Eddinger and associates (43) reported an age-related increase in tetanic tension per unit cross-sectional area of rat soleus muscle.

The cause of the increase in fast fibers and in specific force in the geniohyoid muscle is unclear. In humans, UA resistance increases with age (49); if a similar effect were to occur in rats, this would place an additional load on the geniohyoid and possibly result in a compensatory increase in force. In addition to its role in regulating UA patency, the geniohyoid also has a number of other functions, such as in chewing and swallowing. It is well known that aging is associated with impaired swallowing (50) but, to our knowledge, the effect of age on geniohyoid function during swallowing has not been elucidated. The effects of gender and age on respiratory control and on the regulation of UA patency are poorly understood. It is known that geniohyoid muscle activity is greater in awake, normal women than in men (15). Although the ventilatory response to chemical stimuli has been reported to be attenuated with age in several studies (16)(17)(19), others have reported no significant effects (18)(20).

In conclusion, these results show that gender has no effect on UA muscle contractile properties in either young or old rats. Aging has no effect on sternohyoid muscle contractile function, but it does cause an increase in geniohyoid muscle force production.

Table 1.

Effects of Age and Gender on Geniohyoid Muscle Contractile Properties

 Young  Old  
 Male (n = 21) Female (n = 23) Male (n = 20) Female (n = 20) 
Cross-sectional area (cm20.029 0.027 0.031 0.025 
 (0.003) (0.009) (0.007) (0.006) 
Twitch tension (N·cm−20.73 0.73 0.90 0.70 
 (0.28) (0.45) (0.49) (0.37) 
Tetanic tension (N·cm−22.41 2.49 3.72* 3.56* 
 (1.18) (1.86) (2.0) (1.72) 
Twitch:Tetanic tension 0.27 0.24 0.22 0.22 
 (0.12) (0.12) (0.09) (0.08) 
Contraction time (s) 0.029 0.027 0.031 0.026 
 (0.003) (0.003) (0.005) (0.003) 
Half-relaxation time (s) 0.038 0.035 0.037 0.037 
 (0.007) (0.005) (0.004) (0.004) 
 Young  Old  
 Male (n = 21) Female (n = 23) Male (n = 20) Female (n = 20) 
Cross-sectional area (cm20.029 0.027 0.031 0.025 
 (0.003) (0.009) (0.007) (0.006) 
Twitch tension (N·cm−20.73 0.73 0.90 0.70 
 (0.28) (0.45) (0.49) (0.37) 
Tetanic tension (N·cm−22.41 2.49 3.72* 3.56* 
 (1.18) (1.86) (2.0) (1.72) 
Twitch:Tetanic tension 0.27 0.24 0.22 0.22 
 (0.12) (0.12) (0.09) (0.08) 
Contraction time (s) 0.029 0.027 0.031 0.026 
 (0.003) (0.003) (0.005) (0.003) 
Half-relaxation time (s) 0.038 0.035 0.037 0.037 
 (0.007) (0.005) (0.004) (0.004) 

Notes: Values are means with SD in parentheses. *Indicates significant difference from Young (two-way ANOVA, p < 0.05).

Table 2.

Effects of Age and Gender on Sternohyoid Muscle Contractile Properties

 Young  Old  
 Male (n=21) Female (n=23) Male (n=20) Female (n=20) 
Cross-sectional area (cm20.036 0.036 0.037 0.031 
 (0.007) (0.004) (0.006) (0.005) 
Twitch tension (N·cm−21.38 1.37 1.19 1.01 
 (0.76) (0.73) (0.82) (0.57) 
Tetanic tension (N·cm−24.88 5.04 4.74 5.3 
 (1.89) (1.95) (1.9) (2.7) 
Twitch:Tetanic tension 0.24 0.24 0.25 0.18 
 (0.06) (0.09) (0.15) (0.13) 
Contraction time (s) 0.028 0.028 0.027 0.025 
 (0.003) (0.004) (0.003) (0.002) 
Half-relaxation time (s) 0.040 0.036 0.039 0.037 
 (0.004) (0.008) (0.005) (0.003) 
 Young  Old  
 Male (n=21) Female (n=23) Male (n=20) Female (n=20) 
Cross-sectional area (cm20.036 0.036 0.037 0.031 
 (0.007) (0.004) (0.006) (0.005) 
Twitch tension (N·cm−21.38 1.37 1.19 1.01 
 (0.76) (0.73) (0.82) (0.57) 
Tetanic tension (N·cm−24.88 5.04 4.74 5.3 
 (1.89) (1.95) (1.9) (2.7) 
Twitch:Tetanic tension 0.24 0.24 0.25 0.18 
 (0.06) (0.09) (0.15) (0.13) 
Contraction time (s) 0.028 0.028 0.027 0.025 
 (0.003) (0.004) (0.003) (0.002) 
Half-relaxation time (s) 0.040 0.036 0.039 0.037 
 (0.004) (0.008) (0.005) (0.003) 

Note: Values are means with SD in parentheses.

Figure 1.

Effect of age and gender on geniohyoid muscle contractile properties: A, force-frequency curves for young males (open squares) and females (closed squares); B, force-frequency curves for old males (open circles) and females (closed circles); C, fatigue curves for young males (open squares) and females (closed squares); D, fatigue curves for old males (open circles) and females (closed circles). Bars indicate values expressed as mean ± SD.

Figure 1.

Effect of age and gender on geniohyoid muscle contractile properties: A, force-frequency curves for young males (open squares) and females (closed squares); B, force-frequency curves for old males (open circles) and females (closed circles); C, fatigue curves for young males (open squares) and females (closed squares); D, fatigue curves for old males (open circles) and females (closed circles). Bars indicate values expressed as mean ± SD.

Figure 2.

Effect of age and gender on sternohyoid muscle contractile properties: A, force-frequency curves for young males (open squares) and females (closed squares); B, force-frequency curves for old males (open circles) and females (closed circles); C, fatigue curves for young males (open squares) and females (closed squares); D, fatigue curves for old males (open circles) and females (closed circles). Bars indicate values expressed as mean ± SD.

Figure 2.

Effect of age and gender on sternohyoid muscle contractile properties: A, force-frequency curves for young males (open squares) and females (closed squares); B, force-frequency curves for old males (open circles) and females (closed circles); C, fatigue curves for young males (open squares) and females (closed squares); D, fatigue curves for old males (open circles) and females (closed circles). Bars indicate values expressed as mean ± SD.

This work was supported by a grant from the Royal College of Surgeons in Ireland. We wish to thank T. Dowling and J. Slattery for technical assistance.

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