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Department of Physiology, University of Kentucky, Lexington, Kentucky
Submitted 31 August 2005 ; accepted in final form 19 October 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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larynx; contractile function; fatigue
Because of the multiple roles served by the larynx, its dysfunction compromises both voice quality and airway protection (4, 22, 31, 34). Others have shown that aging alters the structure and properties of the connective tissue in the vocal folds (13, 23). In addition, there is strong evidence of motor denervation of the laryngeal muscles, and a recent study demonstrated that sensory denervation also induces changes in the myosin isoform content of these muscles (9, 16, 30). These extrinsic factors likely interact to exacerbate the effect of aging on the laryngeal muscles. Atrophy of the laryngeal muscles is a common finding in the elderly, and it is a likely cause of idiopathic alterations in voice and swallowing in older patients (20, 22, 31). Abnormal laryngeal kinematics in aged subjects present as impaired adduction of the vocal folds and increased duration of their movements during quiet ventilation; these findings are consistent with weakness of the intrinsic muscles that control the airway entrance (2, 36). To explore the cellular bases of age-related laryngeal dysfunction, the aim of the present study was to measure key indicators of laryngeal muscle function in the Fischer 344 x Brown Norway rat model of aging. The paired thyroarytenoid muscles are adductors laryngeal muscles: each muscle rotates its arytenoid cartilage medially to approximate the vocal folds. These muscles also control bulking, shortening, and tensing of the vocal folds. We used thyroarytenoid muscles from 6-, 18-, and 30-mo-old rats to determine how age alters contractile function and morphology. The experiments tested the hypothesis that rat thyroarytenoid muscles become slower and more fatigable with age.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Use of experimental animals was approved by the Institutional Animal Care and Use Committee and followed the American Physiological Society's Guiding Principles in the Care and Use of Animals. Male Fischer 344 x Brown Norway F1 hybrid rats (6, 18, and 30 mo of age) were obtained from the National Institute on Aging Aged Rodent Colony: we used three rats for each age group for histology and eight rats for each age group for functional studies. The age groups were selected to represent three points in the life span curve of this strain: 6 mo (early flat portion of low mortality), 18 mo (initial increase in mortality), and 30 mo (linear decrease of survival curve) (37). On arrival, the animals were kept in microisolator cages with Harlan Teklad rodent food and water provided ad libitum. Before the collection of tissues, the rats were anesthetized with ketamine and xylazine (80 mg and 12 mg/kg body wt ip) and killed by pneumothorax and exsanguination after a medial thoracotomy. For in vitro function, both thyroarytenoid muscles were dissected intact (including lateral and medial compartments) from origin to insertion, including fragments of thyroid and arytenoid cartilages. For histology, whole larynges were dissected, covered with optimal cutting temperature embedding medium, and frozen in 2-methylbutane cooled to its freezing point in liquid nitrogen.
Histology
Serial 10-µm-thick coronal cryosections of whole larynges were processed concurrently. For overall morphology and mitochondrial content, sections were stained with hematoxylin and eosin and with modified Gomori's trichrome (15, 33). Intracellular glycogen content was estimated with the periodic acid Schiff method (33). After staining, slides were dehydrated in an ethanol series, cleared with xylene, mounted in Permount, and viewed with a Nikon E600 microscope (Nikon, Melville, NY). Images were captured with a Spot RT digital camera (Diagnostic Instruments, Sterling Heights, MI) and a PowerMac G4 computer (Apple Computer, Cupertino, CA) equipped with Spot RT software, version 4.0 (Diagnostic Instruments). Quantitative analyses were done by personnel blinded to the experimental conditions.
Isometric Contractile Function
Both thyroarytenoid muscles were dissected from each larynx (8 rats/age group, 16 muscles/age group) and placed in a tissue bath with platinum field electrodes and filled with a physiological salt solution with the following composition (in mM): 137 NaCl, 5 KCl, 2.0 CaCl2, 1.0 MgSO4, 1.0 Na2HPO4, 24 NaHCO3, 11 glucose, and 0.026 D-tubocurarine, bubbled with 95% O2-5% CO2 to maintain pH at 7.4 at 25°C. The muscles were firmly attached to a force transducer (model AE801, SensoNor, Horten, Norway) and the arm of a servomotor (Aurora Scientific, Aurora, Canada) and stretched to the length giving maximum force in response to electrical stimulation [optimal length (Lo)]. Force and length signals were sampled online and stored for analysis. Measurements included twitch kinetics (force, time to peak force, half relaxation time) and force-stimulation frequency curves, including peak tetanic force (Po). Force was normalized to cross-sectional area.
Velocity of Shortening
We measured speed of shortening with two techniques: load clamp and slack test. The load clamp method determines the force-velocity relationship obtained by isotonic contractions against set loads and estimates shortening velocity (Vmax) as the average speed of all different fiber types present. The slack test determines the unloaded shortening velocity (Vo), an estimate of the shortening velocity of the fastest fibers in the muscle (7, 8, 14). To minimize contractile work before the fatigue protocol, one muscle per animal was used for load clamps and the other muscle for slack tests (8 muscles/age group for each test).
Force-velocity relationship.
Thyroarytenoid muscles were stimulated to contract maximally for 350 ms. In each contraction, the isometric force reached a plateau within 200 ms. The muscle load was changed at 250 ms, and the muscle shortened for the remaining 100 ms. Each muscle contracted against six different loads, applied at 4-min intervals; Po was measured 2 min before each load. The ratios of the applied loads to the preceding maximal force ranged from 
0.2 to 0.7 P/Po (here P is applied load). Shortening velocity was measured as the slope of the linear portion of the length-time curves and plotted with respect to applied load (P/Po). The data were fitted using a least squares method (SigmaPlot, Systat Software, Point Richmond, CA) to a Hill equation describing a rectangular hyperbola (7, 18). Vmax (in Lo/s) is the shortening velocity extrapolated to zero load.
Vo. Thyroarytenoid muscles were stimulated to contract maximally for 350 ms; 250 ms into the stimulation train, the muscle was shortened abruptly, causing force to fall to zero. The time to take up the slack and redevelop force was measured after rapid releases of three different amplitudes (4, 6, and 8% of Lo) performed in consecutive tetani at 2-min intervals. Vo (Lo/s) is the slope of the linear regression line in plots of the release amplitude vs. the time to take up the slack.
Fatigue Protocol
After measurement of all other contractile properties, fatigue was induced by stimulating the muscles (n = 16/age group) at a frequency giving approximately one-half of maximal tetanic force (50–70 Hz) for 500 ms, followed by 1.5-s interval between contractions, until force declined to 50% of the level at time = 0 or for 10 min, whichever occurred first. The fraction of the initial force generated during the last tetanus was used as a fatigue index.
Data Analysis
All results are presented as means (SD). Statistical significance was determined by analysis of variance; post hoc multiple comparisons were done with Student-Newman-Keuls tests (11, 39). The significance level for rejection of the null hypothesis was set at P 
0.05 for all comparisons.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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First, we examined the effect of age on thyroarytenoid muscle fiber size and force production. Figure 1 (left and middle) shows representative micrographs of thyroarytenoid muscles obtained from 6- and 30-mo old rats; there is a noticeable change in fiber size with age. To confirm this observation, Fig. 1 (right) demonstrates that age has a biphasic effect on thyroarytenoid muscle fiber size: mean fiber area doubled from 6 to 18 mo of age, followed by a modest (23%) but significant decrease from 18 to 30 mo. Importantly, fiber size at 30 mo of age was still 59% greater than at 6 mo. The effect of age on isometric contractile properties followed a different pattern. For twitch kinetics, age did not alter time to peak twitch force but increased half relaxation time at 18 and 30 mo of age by just over 30% (Fig. 2A, n = 16 muscles/age). At 30 mo, both twitch and peak tetanic forces were significantly less than at 6 and 18 mo of age (Fig. 2B, n = 16 muscles/age). Although not significantly different, there was also a trend for lower twitch force at 18 mo compared with 6 mo (Fig. 2B).
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The effect of age on the speed of thyroarytenoid muscle shortening was measured by two complementary methods. The load clamp method (Vmax) estimates the average shortening velocity of all fibers within a muscle. Slack tests (Vo) estimate the shortening velocity of the fastest fibers within a muscle. Vmax was not significantly different between muscles from 6- and 18-mo-old rats. By 30 mo, Vmax decreased by 20% compared with 6 mo and was also significantly lower than at 18 mo (Fig. 3A, left; n = 8 muscles/age). Vo decreased steadily with age: compared with 6 mo, it was significantly lower by 19% at 18 mo and by 27% at 30 mo (Fig. 3A, right; n = 8 muscles/age). The age-related decreases in Vmax and Vo did not correlate with gross alterations in myosin ATPase activity. The thyroarytenoid muscles from the three age groups contained 100% type 2 fibers, that is fibers with high histochemical myosin ATPase activity (Fig. 3B). However, many muscle fibers from the 30-mo-old group presented subsarcolemmal areas devoid of myosin ATPase activity that correlated with abnormal mitochondrial clusters as demonstrated with Gomori's trichrome stain (Fig. 3B, left, inset). We did not find equivalent areas in thyroarytenoid muscles from 6- or 18-mo-old animals.
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To examine the effect of age on endurance, thyroarytenoid muscles were induced to fatigue with 500-ms submaximal tetani (50% of peak tetanic force) at 1.5-s intervals until force declined by 50% or for 10 min, whichever occurred first. Muscles were stimulated at 50–70 Hz; modal stimulation frequency was 60 Hz for the 6-mo-old group and 50 Hz for 18- and 30-mo-old groups. Endurance decreased with age. All thyroarytenoid muscles from the 6-mo-old group sustained more than 50% of the initial force for the full 10 min of the fatigue protocol (n = 16 muscles). Only 12 muscles from the 18-mo-old group (75%, n = 16 muscles) and 7 from the 30-mo-old group (44%, n = 16 muscles) maintained more than 50% of initial force for 10 min. At the end of the fatigue protocol, thyroarytenoid muscles from the 6-mo-old group generated 67% of the initial force (n = 16 muscles) compared with 57% (n = 12 muscles) and 52% (n = 7 muscles) for the 18- and 30-mo-old groups, respectively (Fig. 4). The diminished endurance of the muscles from 18- and 30-mo-old groups was associated with histological evidence of age-related shifts in metabolic properties. Muscle sections stained with modified Gomori's trichrome demonstrated that thyroarytenoid muscles from 30-mo-old rats contained fibers with abnormally large mitochondrial accumulations (ragged red fibers), representing 10.4% (SD 4.5) of the muscle fibers. In contrast, ragged red fibers were not found at 6 or 18 mo (Fig. 5). Intracellular glycogen content in the thyroarytenoid muscles changed with age (Fig. 6, A–C). At 6 mo, 7% of muscle fibers showed a positive histochemical reaction for glycogen. The proportion of glycogen-positive muscle fibers significantly increased at 18 mo to 12% and at 30 mo to 27% (Fig. 6D).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Contractile Dysfunction
The thyroarytenoid muscles became weaker and slower with age. Peak twitch and tetanic forces were significantly decreased in the 30-mo-old rats, compared with 6 and 18 mo (Fig. 2B). Together with slower twitch kinetics (increased half relaxation time) and shortening speed (see below), these data suggest that multiple aspects of excitation-contraction coupling and myofilament function are affected by age in the thyroarytenoid muscles. In addition, we found evidence of abnormal structural arrangements that should contribute to the force deficit. For example, there were large mitochondrial accumulations in the muscles from 30-mo-old rats (Figs. 3B and 5). These areas were devoid of myosin ATPase activity and added to the noncontractile intracellular space. In other words, a larger fraction of muscle cross section was occupied by noncontracting material in the 30-mo-old group, decreasing force per unit area. Unexpectedly, there was not clear evidence of muscle atrophy. Whereas mean fiber area decreased from 18 to 30 mo, fiber size at 30 mo was still significantly greater than at 6 mo (Fig. 1). This biphasic pattern of age-related change in muscle fiber size has been described in other muscles; more importantly, some muscles exhibit little or no atrophy in rats of comparable ages (5, 6). Our results indicate that the loss of force-generating capacity is not necessarily associated with overt atrophy in the rat thyroarytenoid muscle. Thirty-month-old Fischer 344 x Brown Norway rats are at the 75% survival stage of their life span curve (37). Given that these animals were not extremely old, it is possible that mean fiber area of the thyroarytenoid muscles could decrease below the 6-mo-old range in rats older than 30 mo.
Vmax of the thyroarytenoid muscles decreased significantly at 30 mo. Vmax represents the average maximal shortening speed of all fibers in a heterogeneous muscle (7, 8). Our data confirm the decrease in Vmax found previously in laryngeal muscles of old baboons (26). The change in Vmax could be due to a shift in the distribution of fiber types present in the thyroarytenoid muscles, for example, an increase in the number of slow fibers. We found no histochemical evidence of age-related changes in myosin ATPase activity (Fig. 3B). Instead, thyroarytenoid muscles of all ages were composed exclusively of fibers with high myosin ATPase activity (i.e., 100% fast type 2 fibers in the three age groups). This finding confirms a recent study that demonstrated that rat thyroarytenoid muscle contains only type 2 fibers, regardless of age (35). In this regard, the thyroarytenoid muscle is different from limb muscles that typically show increases in the relative proportion of type 1 fibers with age (24, 38). Therefore, it is more likely that the age-related decrease in Vmax in thyroarytenoid muscles is due to a shift from faster to slower type 2 fiber types (e.g., from 2B to 2A or 2X), as previously reported (35).
Vo was significantly slower at 18 and 30 mo of age (Fig. 3A). Vo reflects the shortening speed of the fastest fibers in a muscle (7, 8). Others have shown that laryngeal muscles include fibers that express a fast, laryngeal and extraocular muscle-specific myosin heavy chain isoform (12, 29). Muscle fibers containing this myosin isoform would determine Vo, and its potential decrease with age would explain the change in Vo. However, some evidence indicates that this laryngeal muscle myosin heavy chain may not be particularly affected by age (35). If that is the case, then the decrease in Vo could be explained by a change in the relative abundance of slower type 2 fibers, as discussed above for Vmax. This possibility is consistent with the already described age-related shift from 2B to 2X fibers in thyroarytenoid muscles from rats of similar age distribution as in the present study (35).
Loss of Endurance
Rat thyroarytenoid muscles became significantly more fatigable with age. The fatigue protocol that we used for the study (submaximal contractions, 0.25 duty ratio) is intended to last long enough to impose a significant metabolic load on the muscles. For example, fatigable fast hindlimb muscles do not tolerate the full 10-min duration of the protocol, but fatigue-resistant muscles do (28). As anticipated, all thyroarytenoid muscles from the 6-mo-old group completed the 10-min fatigue protocol. In contrast, some thyroarytenoid muscles from 18- and 30-mo-old rats did not sustain more than 50% of the initial force for the duration of the protocol. Moreover, the muscles from the 18- and 30-mo-old groups that completed the fatigue protocol generated significantly less force after 10 min than muscles from the 6-mo-old group (Fig. 4). It should be noted that the stimulation frequency used during the fatigue protocol and the target forces were typically less for the older muscles. Therefore, the effect of age on endurance was probably underestimated. While the immediate cause of contractile failure during the fatigue protocol is multifactorial, two findings suggest that changes in the metabolic profile of the thyroarytenoid muscles from older rats may play an important role in the loss of endurance. First, there was the presence of ragged red fibers in the thyroarytenoid muscles from 30-mo-old rats (Fig. 5). These fibers have abnormal mitochondrial accumulations that give them that characteristic appearance with the Gomori's trichrome stain and are a hallmark of mitochondrial defects associated with aging (1, 5). Our data confirm a recent study that reported abundant ragged red fibers in aging human thyroarytenoid muscle (21). Second, we found an age-related increase in the number of glycogen-positive muscle fibers (Fig. 6). Whether this change represents a compensatory increase in anaerobic pathways in response to abnormal mitochondrial function or an adaptation to altered recruitment patterns remains unknown. In any event, the resulting shift in metabolic characteristics renders the rat thyroarytenoid muscles more fatigable. In other words, the metabolic challenge posed by repetitive contractile activity is no longer tolerated by aging thyroarytenoid muscles.
Significance
The results from this study indicate that intrinsic changes in thyroarytenoid muscle performance and metabolism may be important determinants of abnormal laryngeal function in aged subjects. The muscle weakness and slower contractions demonstrated in vitro are likely to disrupt vocal fold dynamics in the old rats to a significant extent: adaptive changes in motor unit recruitment patterns and timing are probably insufficient to achieve movements of the required amplitude and speed. In consequence, our findings are fully consistent with a novel study in Fischer 344 x Brown Norway rats of similar ages that demonstrated slowing of vocal fold kinematics during quiet breathing (36). Moreover, even compensatory neural activation strategies may be compromised in the elderly: there is strong evidence that aging induces denervation of the intrinsic laryngeal muscles, another factor that would contribute to motor dysfunction in vivo (9, 16). In fact, age-related thyroarytenoid dysfunction must be a multifactorial phenomenon. For example, aging alters the connective tissue in the vocal folds and may change the load imposed on the thyroarytenoid muscle (13, 23). The loss of laryngeal sensory innervation is also likely to induce changes in myosin isoform expression compatible with some of the functional deficits (decreased Vo and Vmax) found in this study (30). Finally, it has been shown in humans that the regenerative capacity of the thyroarytenoid muscle is probably impaired, and it may contribute to age-related fiber loss and atrophy (25). How these extrinsic and intrinsic factors interact to exacerbate the effect of aging on the laryngeal muscles remains unknown. Our data also indicate that aged thyroarytenoid muscles contain abnormal mitochondria and increased glycogen content. These findings are suggestive of diminished aerobic capacity and a shift to increased reliance on glycolytic pathways. This altered metabolic profile correlates with the loss of endurance demonstrated in our in vitro studies, negatively impacting activities requiring sustained glottal closure and/or tensioning such as swallowing and airway protective reflexes (cough, sneeze).
In conclusion, our data support the initial hypothesis that the thyroarytenoid muscles of Fischer 344 x Brown Norway F1 hybrid rats become weaker, slower, and more fatigable with age. We also found evidence suggestive of abnormal mitochondrial function and a greater reliance on anaerobic glycolytic pathways in the thyroarytenoid muscles from old rats. These data provide an initial correlation between loss of laryngeal muscle endurance and age-related metabolic dysfunction. Therefore, aging induces important functional alterations in the intrinsic laryngeal muscles that may be of great clinical relevance in the elderly.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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