HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Norton, M. W. Articles by McCarter, R. J.M. Search for Related Content PubMed PubMed Citation Articles by Norton, M. W. Articles by McCarter, R. J.M.

Age, Fatigue, and Excitation-Contraction Coupling in Masseter Muscles of Rats

^a Department of Physiology, University of Texas Health Science Center, San Antonio

Roger J.M. McCarter, Department of Physiology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284 E-mail: mccarter{at}uthscsa.edu.

Decision Editor: John A. Faulkner, PhD

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The purpose of this study was to determine if masseter muscle endurance changes with increasing age and, if so, to examine mechanisms of fatigue. Characteristics of fatigue were measured under isometric conditions using high-frequency stimulation of anterior deep masseter (ADM) muscles of male Fischer 344 rats, 5 to 24 months old, and fed a hard (HD) or a soft (SD) diet. Potentiating effects of caffeine on ADM muscle performance in vitro were also examined. Fatigability increased by 48% with age in muscles of HD rats. Muscles of SD rats were highly fatigable at all ages. Increased HD fatigability was associated with significantly decreased concentrations of Na⁺/K⁺-adenosine triphosphatase (22%) and decreased responsiveness to caffeine postfatigue (29%). The pH levels decreased similarly in fatigued muscles of all groups. We conclude that the age-related increase in fatigability is associated with alterations in excitation-contraction coupling mechanisms. However, differences between SD and HD on ADM muscles represent possible fiber-type transitions.

DECREASED physical activity and loss of muscle mass are characteristic features of advancing age ⁽¹⁾⁽²⁾. Mechanisms involved in this loss have not been identified, but decreased physical activity is widely believed to be an important contributing factor. Associated with this decrease in skeletal muscle mass is also a decrease in endurance characteristics in elderly persons ⁽³⁾. With regard to the muscles of mastication, investigation of the mechanistic basis of these effects has been hampered by the absence of a suitable animal model, because studies involving maximum isometric contractions in vivo elicit pain and fatigue in healthy individuals ⁽⁴⁾. However, masseter muscles are too thick to survive in vitro. We have therefore identified a branch of this muscle that is sufficiently thin in Fischer 344 (F344) rats to survive in vitro and to maintain function for several hours. This branch, the anterior deep masseter (ADM) muscle, has a similar fiber composition to that of the whole masseter muscle. Using this preparation in vitro we have demonstrated that muscle-twitch characteristics and specific tension-generating capabilities of the ADM muscles of male F344 rats do not decline with increasing age ^(5)(6)(7).

Muscle fatigue in elderly persons may be the result of alterations in several different mechanisms. Traditionally, fatigue has been defined as the failure to maintain a required or expected force ⁽⁸⁾. However, causes of fatigue probably involve several factors and multiple sites along the path of muscle activation, from the central nervous system to the myosin cross bridges of the thick filaments of muscle fiber ^(9)(10). Masticatory muscle function is more complex than that of limb skeletal muscle because it is affected by sensory information regarding pressure and noxious stimuli at the teeth and gums ⁽¹¹⁾. Because of its inherent complexity, it is necessary to isolate those aspects of mastication that might reflect only aging of muscle tissue through the use of muscle preparations in vitro. The goal of the present investigation was to determine if the endurance of rat masseter muscles changes with age and, if so, to investigate mechanisms of altered fatigability. In particular, we tested the hypothesis that masseter muscles of rats are more fatigable with advancing age and that this increase in fatigue is associated with compromised mechanisms of excitation-contraction (E-C) coupling. Given the well-known preference of elderly persons for soft-textured foods ^(12)(13)(14) and the equally well established effects of decreased activity on muscle function ⁽²⁾, a further goal of this study was to characterize the effects on endurance of a soft-texture diet.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Animals
Male specific-pathogen–free F344 rats were obtained from two sources: the National Institute on Aging (NIA) colony (Harlan Sprague Dawley, Inc., Indianapolis, IN) and the barrier facility of the Aging Research Group of the University of Texas Health Science Center in San Antonio (UTHSCSA; Department of Physiology). They were maintained in a specific-pathogen–free environment with a light/dark cycle of 12/12 hours until the time of sacrifice. All rats had access to food and water ad libitum. The F344 rat was chosen for this study because its age-related physiological and pathological characteristics have been well established and extensively documented ⁽¹⁵⁾. Masseter muscles from healthy F344 rats of 4 to 6, 12, 18, and 24 months of age purchased from the NIA were used for determination of masseter muscle endurance. These animals were fed a standard hard-pellet rat chow (Teklad NIH-31). Mechanisms involved in fatigability were investigated in rats, 4 to 6 and 24 months old, fed ad libitum from this source.

For the determination of the effects of altered workload on masseter muscle function, we used male specific-pathogen–free F344 rats (4–6, 12, and 24 mo) that had been maintained in the barrier facility of the UTHSCSA Aging Research Group. These barrier-raised animals were fed powdered rat chow from the time of weaning (3–4 weeks old) onwards (Purina SR Vitamin-fortified RP 101). Rats were sacrificed the same day as removal from the barrier facility. Although the compositions of the hard-pellet diet (HD) and the soft-powdered diet (SD) feed were slightly different, these differences previously have been shown to have no significant effect on muscle fiber composition in F344 rats ⁽¹⁶⁾.

Masseter Muscle Preparation
Animals were weighed and then anesthetized with methoxyflurane prior to sacrifice by decapitation. ADM muscles were carefully dissected bilaterally using a dissecting microscope to reveal an origin at the rostral maxillary portion of the zygomatic arch and an insertion on the masseteric ridge of the mandible. Muscles were placed in oxygenated (95% O₂ and 5% CO₂) pH 7.4 Ringer's solution (in mmol/l: 115.0 NaCl, 5.0 KCl, 0.65 MgSO₄, Na₂H₂PO₄, 25.0 NaHCO₃, 2.5 CaCl₂, 11.0 glucose, and 0.021 d-tubocurarine chloride), except for those specimens collected for potassium contracture experiments. Each muscle preparation was carefully trimmed of damaged fibers to a width of approximately 2 to 3 mm. Stainless steel rings (size 0 surgical wire, Ethicon, Somerville, NJ) were tied with silk thread to the maxillary bony fragment at one end and the mandibular myotendinous junction at the other end. This preparation was used for determination of contractile properties, caffeine responses, high-potassium responses, and pH measurements as described later. For the determination of Na⁺/K⁺-adenosine triphosphatase (ATPase) content, the bony attachment sites of the ADM were removed following microscopic dissection. These preparations were then frozen in liquid nitrogen and stored at ^-80°C until used for the determination of Na⁺/K⁺-ATPase.

Measurement of Muscle Contractile Properties
Muscle preparations were mounted in a horizontal muscle bath (40-ml volume) between two parallel platinum-plate electrodes. The muscle bath contained curarized, oxygenated Ringer's solution at room temperature (23°C). The maxillary end of the ADM preparations was connected to a fixed rod and the mandibular end to a force transducer (model 352, Cambridge Technology, Inc, Aurora Scientific, Richmond Hill, Ontario, Canada). Isometric properties were measured with the muscle held at a fixed length in response to supramaximal stimulation. Output of the force transducer was displayed on a computer screen using an analog to digital converting system and compatible software (Scope V3.6, AD Instruments, Inc, Mountain View, CA). A Grass S88 AstroMed, Inc., Warwick, RI stimulator in series with a stereo power amplifier (ADCOM 2200, East Brunswick, NJ) provided supramaximal electrical pulses of a 0.25-millisecond duration to the parallel electrodes surrounding the muscle preparations. For development of maximal tetanic tension, muscles were stimulated at a frequency of 80 Hz and a train duration of 500 milliseconds at optimal length (L_o). L_o was defined as the length at which the muscle developed maximal twitch (P_t) and maximal tetanic (P_o) tension. L_o was measured for each preparation to a precision of ±0.1 mm with a micrometer eyepiece mounted directly above the preparation. All preparations were stimulated at 10 Hz above and 10 Hz below 80 Hz to verify the optimal stimulation frequency. An index of fatigue was determined by stimulating a muscle preparation ten times every 30 seconds for 8 seconds, at a frequency of 80 Hz. Maximal tetanic tension obtained in the tenth tetanus divided by maximal tension obtained in the first tetanus was defined as the index of fatigue (F_i). A muscle that has high fatigability would therefore be indicated by an F_i approaching 0, whereas an F_i close to 1.0 would indicate a more fatigue-resistant muscle. Following every fatigue protocol, muscle preparations were allowed to recover for 5 minutes in oxygenated (95% O₂ and 5% CO₂) pH 7.4 Ringer's solution. After a 5-minute recovery period, muscle preparations were supramaximally stimulated to determine recovery isometric twitch and tetanic tension.

Stability of the Preparation In Vitro
Maximum isometric tension of all muscle preparations, measured at the start of each experiment, varied from 21 to 28 N/cm², well within the range of performance expected for healthy mammalian skeletal muscle ⁽¹⁷⁾. Experiments typically had a duration of 1 hour, commencing approximately 20 minutes after termination of the animal. In control experiments, with tension measured every 10 minutes, the maximum tension declined less than 10% over a period of 2 hours. We are thus confident that these preparations from rats of all ages consisted of intact muscle fibers that remained viable throughout the course of each experiment.

In addition, following the strenuous high-frequency fatigue protocol used in this study, recovery tension in randomly chosen ADM preparations from all experimental SD and HD groups (n = 3) displayed a mean percentage of P_o recovery of 90% ± 7%, by 30 minutes postfatigue. These results confirm the remarkable viability of this mammalian muscle preparation over several hours in vitro.

Determination of Na⁺/K⁺-ATPase Concentrations
Quantification of the concentration of Na⁺/K⁺-ATPase in muscles was determined by measurements of bound [³H]oubain as described by Notgaard and colleagues, Kjeldsen, and Green and colleagues ^(18)(19)(20). Following these methods, muscles that had not been subjected to any other experimental procedures were removed and kept frozen at -80°C until the time of assay. Each muscle sample was then cut into three portions, each weighing 2 to 8 mg. These portions were placed into glass tubes for two 10-minute prewashes at 0°C, with continuous gassing to ensure agitation. The prewash was a tris-sucrose buffer containing vanadate (in mmol/l: 10 Tris HCl, 1.0 MgSO₄, 250 sucrose, and 1.0 vanadate; pH 7.3). Vanadate was utilized in this technique because, in the absence of ATP, it facilitates the binding of [³H]oubain to the Na⁺/K⁺-ATPase ⁽²¹⁾. Samples were then incubated in the same buffer with the addition of [³H]oubain (2.0 µCi) and unlabeled oubain (5 x 10^-6 mol/l final concentration) for 30 minutes at 37°C. After 30 minutes, samples were transferred to fresh [³H]oubain buffer and allowed to incubate an additional 30 minutes to allow [³H]oubain distribution in tissue samples to reach a steady state ⁽¹⁹⁾. Unbound oubain was then removed by washing the samples four times, for 30 minutes each, in the previously described ice-cold buffer. Next, samples were blotted and weighed. Samples underwent 16 hours of extraction in 0.5 ml of 5% trichloroacetic acid at room temperature (23°C). Scintillation cocktail was added to the vials prior to measurement of [³H] radioactivity. The isotopic purity was 96% to 99%, as determined by the supplier (Amersham-Buckinghamshire, UK) using chromatographic techniques. Unspecific retention and/or binding was determined in each experiment by running a set of samples in hot incubation buffer that contained an excess (10^-3 mol/l) of unlabeled oubain. Bound ³H radioactivity was calculated and expressed as relative uptake (ml/g of wet-muscle weight). Relative uptake of ³H radioactivity (minus relative uptake of ³H radioactivity representing unspecific binding) was then multiplied by the concentration of oubain in the incubation medium to express specific activity (i.e., binding of oubain to Na⁺/K⁺ pumps per unit of muscle mass) in pmol/g wet-weight of muscle.

Potassium Contractures
Maximum K⁺ contracture tension was elicited using a method similar to that of Cairns and Dulhunty ⁽²²⁾. Bilateral ADM muscles were removed from each experimental animal. Fine dissection of the ADM muscle was performed in the low chloride ion [Cl^-] solutions, and preparations equilibrated additional time to allow a total of 1-hour equilibration in oxygenated low [Cl^-] solutions. Low [Cl^-] solutions were a modified Krebs solution containing the following (in mmol/l): 2.0 N-tris [hydroxylmethyl] methyl-2-aminoethanesulfonic acid; 2-([2-hydroxy-1, 1-tris (hydroxy methyl)-ethyl]amino) ethane sulfonic acid (TES), 1.0 MgSO₄, 40.25 Na₂SO₄, 1.75 K₂SO_4, 170 sucrose, 11 glucose, and 0.021 curare; pH 7.4. L_o, P_t , and P_o were determined for every muscle preparation in a low [Cl^-] bathing medium. One side was used to evaluate prefatigue K⁺ contracture tension. The contralateral side was used to evaluate postfatigue contracture tension. K⁺ contractures were elicited with a 200 mmol/l [K⁺] oxygenated solution warmed to 37°C, which contained the following (in mmol/l): 100 K₂SO₄, 8 CaCl₂, 2.0 TES, 1.0 MgSO₄, 11 glucose, and .021 curare; pH 7.4. Pre- and postfatigue contracture tensions are reported as a fraction of P_t and P_o.

Caffeine Treatment
Both ADM muscles were removed from each experimental animal. All muscle preparations were dissected and maintained at room temperature (23°C) in curarized, oxygenated Ringer's solution. L_o, P_t , and P_o were established for all preparations before treatment with caffeine. One ADM muscle was used to determine the potentiating effects of caffeine on maximum twitch and tetanic tension without establishing F_i. Contralateral ADM muscles were used to determine the effects of caffeine on 5-minute recovery of twitch and tetanus tension after determining F_i. Caffeine treatment consisted of a 5-minute exposure to a 37°C Ringer's solution with the addition of a 32-mmol/l concentration of caffeine. Measurements of contractile properties were determined using stimulation parameters as previously described. Pre- and postfatigue caffeine potentiations are reported as percentages of P_t and P_o obtained in caffeine-free Ringer's solution at the start of the experiment.

pH Measurements
Cellular responses of muscle preparations to fatigue were monitored by using a muscle homogenate technique that gives a reliable estimate of average intracellular pH (pH_i) ^(23)(24)(25). Resultant measurements are not significantly different from other commonly used techniques for determining pH_i ^{(23)(24)(25)(26)(27)}. Bilateral ADM muscles were dissected and prepared for determination of contractile properties as described previously. Contractile properties were determined for one preparation. Within the first 20 seconds following the determination of F_i, the muscle preparation was removed from the muscle bath; the metal rings and bone were cut from the preparation; and the preparation was blotted dry and frozen in liquid nitrogen. The contralateral preparation was maintained in oxygenated Ringer's solution for the duration of the experiment at which time it too was frozen in liquid nitrogen. Samples were then freeze dried, dissected free of connective tissue, and powdered. A portion of the sample (1.5–2 mg) was homogenized at 0°C in a solution containing 145-mmol/l KCl, 10-mmol/l NaCl, and 10-mmol/l NaFl (pH 7.2), at a dilution of 30 mg of dry muscle weight per ml of homogenizing solution. NaFl was added to prevent further glycolytic reactions during handling. pH measurements were then made using a microelectrode connected to a pH/ion analyzer (model M1-415, Microelectrode, Inc, Bedford, NH), as described by Mannion and colleagues ⁽²⁴⁾. The response of the ADM muscle pH_i to fatigue was calculated as the difference in pH between the ipsilateral fatigued ADM and the contralateral nonfatigued ADM from the same experimental animal.

Data Analysis
A one-way analysis of variance (ANOVA) was used to analyze HD experimental data and SD experimental data separately. Tukey-Kramer multiple comparisons tests were used to determine if changes in the independent variable (age of rat) caused significant differences in the dependent variables (fatigue index and percentage of tetanic tension recovery), first within the HD and then within the SD group. Significance was assumed at p < .05. For fatigue-index data, a lack-of-fit test was used to determine the appropriateness of a straight-line model for both HD and SD group data. Resultant p values (together with the absence of any overall significant difference with age in the SD group) supported the use of straight-line models for both HD and SD fatigue data. Significance was assumed at p < .05. Both HD and SD age groups were then combined and analyzed using a one-way ANOVA for the fatigue-index dependent variable. The Tukey-Kramer multiple comparisons test was used to test for significant differences ( p < .05) among all groups ⁽²⁸⁾. Bonferroni adjustments to the Tukey-Kramer multiple comparisons tests between the 5-month HD and 5-month SD, and between the 5-month HD and 24-month HD, assumed significant differences at p < .025.

All other statistical comparisons consisted of one-way ANOVA analysis of 5-month HD versus 24-month HD groups (age = independent variable). In addition, one-way ANOVA was used to analyze 5-month HD versus 5-month SD groups (diet = independent variable). Unequal variances were further analyzed using Levene and Bartlett's tests ( p < .05). Student t tests were used to detect significant difference at p < .05.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Fatigue and Recovery
Data on fatigue of HD-ADM muscle preparations are presented in Table 1 . An age-related decline in ADM muscle F_i was found. F_i in 18- and 24-month-old rats (0.13 ± 0.02 and 0.11 ± .02, respectively) was less than in 12-month F_i responses (0.19 ± 0.04) and significantly less than in the F_i responses of the 5-month group (0.21 ± 0.02). Linear regression analysis of these data revealed an r² = .945. These data indicate a significant age-related decline in endurance of masseter muscles. Recovery tensions are presented in Table 1 as a percentage of the maximum tetanic tension obtained prior to fatigue. There was no significant difference in recovery tetanic tension among the age groups studied. In addition, recovery twitch tensions measured in young and old HD groups were not significantly different.

F_i from muscles of young HD and SD animals and from old HD and SD animals are shown in Fig. 1. This figure depicts the age-related changes in fatigability found in HD-ADM preparations combined with SD-ADM preparations. ADM muscles from young HD rats were found to be significantly less fatigable than their young SD counterparts. Moreover, all SD-ADM muscles were significantly more fatigable than young HD-ADM muscles and had fatigability similar to that of old HD-ADM muscles.

View larger version (31K): [in this window] [in a new window]   Figure 1. Fatigue index in the anterior deep masseter muscle as a function of age and diet: ratio of tetanic tension developed in the 10th isometric tetanus to tension developed in the 1st isometric tetanus for muscles from rats of two different ages fed a hard or a soft diet. Values are means ± SE. For all groups: n = 10, except 24-month soft-diet group (n = 4). *Significantly different group, p < .05.

View larger version (47K): [in this window] [in a new window]   Figure 2. Effect of age on concentration of Na+/K+-ATPase in hard diet–anterior deep masseter muscles. Data from binding of radioactive oubain to Na+/K+-ATPase pumps in muscle homogenates from rats of two different ages. Bar graph represents pmol of [3H]oubain binding per gram of wet muscle weight. Values are means ± SE. For 5-month group, n = 13; for 24-month group, n = 12. *Significantly different group, p < .05.

Effects of High-Potassium Solutions
Table 2 shows data gained from exposing ADM muscles to Ringer's solutions containing high concentrations of K⁺. Contracture tensions induced by this method of membrane depolarization were normalized to peak twitch and peak tetanic tension elicited by electrical stimulation and recorded prior to exposure to high potassium solutions. Postfatigue twitch and tetanic tension was significantly less than prefatigue tension in both experimental HD groups. There were no significant differences in pre- to postcontracture tension changes between young and old groups (i.e., membrane responses to depolarization were similar in young and old HD-ADM muscles).

Effects of Caffeine Potentiation
Table 3 contains data from caffeine potentiation experiments. Responses are reported as percentages of peak twitch and tetanic tensions. Young and old HD-ADM showed significantly greater twitch responses after exposure to a solution of 32-mmol/l caffeine than precaffeine twitch responses. There was no significant difference between net potentiation in twitch responses of old and young groups. There were no significant potentiating effects of caffeine on maximum tetanic tensions of either young or old ADM muscle preparations. This confirms that the supramaximal stimulation parameters were sufficient to elicit maximal calcium release when characterizing ADM isometric contractile properties.

Young SD-ADM muscle exhibited significantly greater twitch responses when exposed to 32-mmol/l caffeine in unfatigued muscles (data not shown; t test, p < .05). But, there was no significant difference in net potentiation of twitch responses between SD-ADM and HD-ADM muscles. Again, caffeine had no significant potentiating effects on maximum tetanic tensions of SD- or HD-ADM muscle preparations (Table 3 ).

Recovery twitch and tetanic tension responses were significantly greater in ADM muscles that had been treated with caffeine, compared with noncaffeine treatment in 5-month-old SD rats. However, when young SD- and young HD-ADM muscles were compared, caffeine did not have significantly different postfatigue potentiating effects.

pH Measurements
Table 4 presents results of pH_i measurements of HD rats. Data reported are pH_i prefatigue and pH_i postfatigue. The pre- to postfatigue decreases in pH_i are reported as mean change in pH_i. There was a significant decrease in pH_i due to fatigue of ADM muscles in both young and old HD animals. However, there was no age-related difference in the mean change in pH_i.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Previous work in our laboratory found that ADM muscles do not display age-related decline in composition and acute performance. However, a major finding of this study is that endurance decreased with increasing age in ADM muscles of F344 rats in a linear fashion. Also, a diet of soft consistency resulted in highly fatigable masseter muscles at any age. It appears that neither aging nor decreased activity due to a diet of soft consistency had a detrimental effect on the ability of the contractile proteins of the ADM muscle to develop isometric tension under optimal conditions in vitro. But, when these same muscle preparations were subjected to repetitive high-frequency stimulation, as in the present fatigue protocol, differences in fatigability were found. This is worth emphasizing because this type of fatigue has been shown to depend primarily on ionic changes ⁽¹⁰⁾. Also, the rate and development of high-frequency fatigue involves several factors that are pertinent to mechanisms of E-C coupling. The actions of the Na⁺/K⁺ pump are essential to the restoration and maintenance of muscle membrane excitability following repeated activity. The significant decrease in [³H]oubain binding found with age in ADM muscles suggests that compromised ion-pumping capabilities may play a role in the development of high-frequency fatigue in the old HD-ADM muscle. Decreased concentrations of Na⁺/K⁺ pumps located along the surface of the sarcolemmal membrane might affect the propagation or the characteristics of surface membrane action potentials during such fatiguing protocols ^(29)(30). In whole-muscle electrophysiology, the M wave is the peak-to-peak amplitude of a compound muscle action potential ⁽²⁹⁾. Its amplitude depends on both the resting membrane potential and the extent of overshoot of the action potential of single muscle fibers. Significant decrements in the size of the M waves of several limb muscles in elderly persons have been found ^{(29)(30)(31)(32)}. Hicks and colleagues ⁽²⁹⁾ proposed that these data are indicative of decreased activity of Na⁺/K⁺-ATPase pumps. These decrements in M waves could represent decreases in Na⁺/K⁺-ATPase pump density similar to those found in this study. In particular, our study suggests that similar decrements in M-wave amplitude would be found in masseter muscles of elderly men and women.

The concentration of Na⁺/K⁺-ATPase pumps in skeletal muscle is affected by a number of physiological factors. Among these are developmental changes ⁽³³⁾, thyroid hormone (T₃) levels ⁽³⁴⁾, and increased activity as well as decreased activity ^(20)(34)(35). Although numerous studies on rodents and humans show trends toward decreases in [³H]oubain binding with increasing age in various skeletal muscles, none have shown significant declines. The results of the present study are important because they show an age-associated decline in Na⁺/K⁺-ATPase pumps with increasing age in the ADM muscle. Yet, food consumption measurements in our laboratory showed no significant difference in food consumed per day between 5- and 24-month-old HD rats (data not shown). This suggests that decreased masseter muscle activity was not responsible for the decline in functional activity of Na⁺/K⁺-ATPase. And, recent investigations on thyroid hormone levels in aged rodents have revealed no significant decrease in T₃ levels (biologically active hormone) with increasing age. However, current research points to a decreased responsiveness of various tissues in aging F344 rats to thyroid hormone ^(36)(37). Studies also show that epinephrine and norepinephrine stimulate active Na⁺-K⁺ transport in muscle cells by 100% ^(38)(39)(40). Moreover, this increased Na⁺-K⁺ transport in muscle cells occurs at physiological concentrations of these catecholamines ^(38)(39). Thyroid hormone is the major endocrine factor responsible for the synthesis of proteins associated with the Na⁺/K⁺-ATPase pump. These facts, taken together with data gained from the results of the current investigation, suggest the possibility of decreased sensitivity of aging ADM muscles of the F344 rats to thyroid hormone stimulation and concomitant decline in Na⁺/K⁺-ATPase concentrations. The correlation between increased fatigability and decreased Na⁺/K⁺-ATPase pump concentration suggests that there is a decreased ion-pumping capacity in aging F344 ADM muscles. This loss of pumping capacity is not manifested as a decrease in aged ADM muscle performance under normal brief physiological conditions because the usual safety factor of redundant Na⁺/K⁺ pumps, so often found in biological systems, safeguards against this. However, it does reflect an age-related loss of potential for sustained activity of the ADM under extreme conditions.

It is well established that the dihydropyridine receptors (DHP) located in the transverse (T-) tubules of the sarcolemma are involved in E-C coupling as voltage sensors. In the experiments conducted in this study, exposure of ADM muscles to high extracellular potassium solutions caused the DHP receptor to sense a change in membrane potential. Subsequent calcium release from the sarcoplasmic reticulum (SR) caused the development of tension known as contracture tension. Prefatigue and postfatigue contractures were not different when young and old ADM muscles were compared. These data thus support the conclusion that there is no difference in response of the voltage sensors to artificial depolarization, regardless of animal age or state of fatigue.

The use of caffeine allows the investigator to bypass depolarization-induced release of calcium from the SR during E-C coupling and to focus on the release of caffeine-sensitive pools of calcium through the ryanodine receptor calcium channels of the SR. Present results demonstrated that there was no difference in the potentiating effects of caffeine on twitch responses of the young and old ADM muscles. This indicates that, under physiological conditions, calcium handling was as effective in the aged ADM as in young ADM muscles. However, after submitting these muscle preparations to the high-frequency fatigue protocol, and allowing 5 minutes recovery in oxygenated caffeine solutions, differences emerged. The potentiating effects of caffeine on recovery tension were significantly less in aged muscles when compared with young muscles. Hence, under the conditions created by 80-Hz stimulation, calcium reuptake or release appeared to be compromised in fatigued aging muscle. There are several ways that resequestration could be affected: a small pool of Ca⁺¹ might accumulate in the T-tubule ⁽⁴¹⁾; the concentration of the cytosolic Ca⁺¹-binding protein, parvalbumin, might be increased; finally, and most likely, the activity of the SR re-uptake pump, Ca⁺¹-ATPase, might be reduced in the aging ADM. Increased age-related fatigability would then reflect a compromised re-uptake or release of calcium by the SR in ADM muscles, although evidence on this point is ambiguous ⁽¹⁾.

Finally, a major metabolic byproduct that accumulates in the myoplasm during sustained contractile activity is the hydrogen ion. It has been shown that increases in intracellular hydrogen ions ([H⁺]_i) cause decreases in Ca⁺¹ sensitivity of the myofilaments in single-fiber experiments ⁽⁴²⁾. Fitts ⁽⁹⁾ has postulated that free H⁺ may also inhibit Ca⁺¹ binding to the DHP receptor of the T-tubule. Ultimately, this would result in a reduced calcium transient and perhaps loss of tension during fatiguing stimulation of skeletal muscles. Any or all of these phenomena may occur during the high-frequency fatigue protocol of the ADM muscles. However, data from the current study argue against this theory as an explanation for changes in ADM muscle with age. Evidently, the higher fatigability of aged ADM muscles when compared with young ADM muscles is not the result of a greater increase in myoplasmic acidity.

Central to any discussion involving aging skeletal muscle is the issue of whether structural and/or functional changes are the consequence of muscular inactivity. Therefore, it is important to differentiate between aging and the effects of decreased activity per se on the muscle. With regard to the masseter muscle, animals fed an SD provide a model of decreased chewing activity, since there is no need to break up pellets of food. In the current study, significant differences in responses were elicited during steps of E-C coupling in the aging model but not in the altered activity model. Hence, it can be concluded that age and altered activity represent different structural or compositional changes in the experimental muscles. To return to the hypothesis of this study, the present investigation demonstrated that masseter muscles of rats are more fatigable with advancing age and that this increase in fatigability is associated with compromised mechanisms of E-C coupling. Specifically, this age-related increase in fatigability was associated with a decrease in Na⁺/K⁺-ATPase concentration and a decrease in release of caffeine-sensitive SR calcium pools by fatigued muscles. Eating an SD versus an HD was associated with an increase in fatigability, but this did not increase with age. It is possible that differences between SD- and HD-ADM muscles represent fiber-type transitions. Future research should be directed toward detailed analysis of intermediate muscle-fiber types, investigations of Na⁺/K⁺-ATPase concentration in T-tubular membranes and investigations of calcium transients in the aged and altered activity models of ADM muscles.

	Acknowledgments

 
This work was supported by the National Institutes of Health Grant P50 DE 10756.

Received November 26, 1999

Accepted August 24, 2000

	References

Top Abstract Materials and Methods Results Discussion References

 

1. McCarter RJM, 1990. Age-related changes in skeletal muscle function. Aging. 2:27-38. [Medline]
2. Fiatorone MA, Evans WJ, 1993. The etiology and reversibility of muscle dysfunction with the aged. J Gerontol. 48: (special issue) 77-83.
3. Faulkner JA, Brooks SV, Zerba E, 1990. Skeletal muscle weakness and fatigue in old age: underlying mechanisms. Ann Rev Gerontol Geriatr. 10:147-166. [Medline]
4. Jow RW, Clark GT, 1989. Endurance and recovery from sustained isometric contractions in human jaw-elevating muscles. Arch Oral Biol. 34:857-862. [Medline]
5. Norton MW, Versteegden AW, Maxwell LC, McCarter RJ, 1995. Effects of age on structure and function of rat masseter muscle. FASEB J. 9:A651
6. Versteegden AWP, 1995[master's thesis]. Characterization of Age-Related Changes in Masseter Muscle of Male and Female Fischer 344 Rats: A Histochemical Study Rijksuniverstiteit Limburg, Maastricht.
7. Norton MW, 1998[dissertation]. Characterization of Rat Masseter Muscle Function with Age University of Texas Health Science Center, San Antonio, TX.
8. Edwards RHT, 1981. Human muscle function and fatigue. Porter R, Whelan J, , ed.Human Muscle Fatigue: Physiological Mechanisms 1-20. Pitman, London.
9. Fitts RH, 1994. Cellular mechanisms of muscle fatigue. Physiol Rev. 74:49-94. [Abstract/Free Full Text]
10. Westerblad H, Lee JA, Lannergen J, Allen DG, 1991. Cellular mechanisms of fatigue in skeletal muscle. Am J Physiol. 261:C195-C209. [Abstract/Free Full Text]
11. Bakke M, 1993. Mandibular elevator muscles: physiology, action and effect of dental occlusion. Scand J Dent Res. 101:314-331. [Medline]
12. Idowu AT, Graser GN, Handelman SL, 1986. The effect of age and dentition status on masticatory function in older adults. Special Care Dent. 6:80-83.
13. Sandstrom B, Lindquist LW, 1987. The effect of different prosthetic restorations on the dietary selection of edentulous patients. Acta Odontol Scand. 45:423-428. [Medline]
14. Hartsook E, 1974. Food selection, dietary adequacy and related dental problems of patients with dental prostheses. J Prosthet Dent. 32:32-40. [Medline]
15. Yu BP, Masoro EJ, McMahon CA, 1985. Nutritional influences on aging of F344 rats. I. Physical metabolic and longevity characteristics. J Gerontol. 40:651-670.
16. McCarter R, McGee J, 1987. Influence of nutrition and aging on the composition and function of rat skeletal muscle. J Gerontol 42:423-441. [Medline]
17. Close RI, 1972. Dynamic properties of mammalian skeletal muscles. Physiol Rev. 56:129-197.
18. Norgaard A, Kjeldsen K, Hansen O, Clausen T, 1983. A simple and rapid determination of the number of 3H-ouabain binding sites in biopsies of skeletal muscle. Biochem Biophys Res Commun. 111:319-325. [Medline]
19. Kjeldsen K, 1986. Complete quantification of the total concentration of rat skeletal-muscle Na+/K+-dependent ATPase by measurements of [3H]oubain binding. Biochem J. 240:725-730. [Medline]
20. Green HJ, Chin ER, Ball-Burnett M, Ranney D, 1993. Increase in human skeletal muscle Na+/K+-ATPase concentration with short-term training. Am J Physiol. 264:C1538-C1541. [Abstract/Free Full Text]
21. Hansen O, 1979. Facilitation of oubain binding to Na⁺/K⁺-ATPase by vanadate at in vivo concentrations. Biochim Biophys Acta. 566:265-269.
22. Cairns SP, Dulhunty AF, 1995. High-frequency fatigue in rat skeletal muscle: role of extracellular ion concentrations. Muscle Nerve. 18:890-898. [Medline]
23. Costill DL, Sharp RL, Wink WJ, Katz A, 1982. Determination of human muscle pH in needle-biopsy specimens. J Appl Physiol. 53:1310-1313. [Abstract/Free Full Text]
24. Mannion AF, Jakeman PM, Willian PM, 1995. Skeletal muscle fiber value, fiber type distribution and high intensity exercise performance. Exp Physiol. 80:89-101. [Abstract]
25. Sullivan MJ, Saltin B, Negro-Vilar R, Duscha BD, Charles HC, 1994. Skeletal muscle pH assessed by biochemical ³¹P-MRS methods during exercise and recovery in men. J Appl Physiol. 77:2194-2200. [Abstract/Free Full Text]
26. Boron WF, Roos A, 1976. Comparison of microelectrode, DMO, and methylamine methods for measuring intracellular pH. Am J Physiol. 231:799-809. [Abstract/Free Full Text]
27. Porter M, Boutilier RG, 1990. Determination of intracellular pH and P_CO2 after metabolic inhibition by fluoride and nitrilotriacetic acid. Respir Physiol. 81:255-274. [Medline]
28. Zar JH, 1984. Multiple comparisons. Zar JH, , ed.Biostatistical Analysis 185-205. Prentice Hall, Englewood Cliffs, NJ.
29. Hicks AL, Cupido CM, Martin J, Dent J, 1992. Muscle excitation in elderly adults: the effects of training. Muscle Nerve. 15:87-93. [Medline]
30. Sjogaard G, 1990. Exercise-induced muscle fatigue: the significance of potassium. Acta Physiol Scand. 593:1-63.
31. Vandervoort AA, McComas AJ, 1986. Contractile changes in opposing muscles of the human ankle joint with aging. J Appl Physiol. 61:361-367. [Abstract/Free Full Text]
32. Campbell MJ, McComas AJ, Petito F, 1973. Physiological changes in ageing muscles. J Neurol Neurosurg Psychiatry. 36:174-182. [Abstract/Free Full Text]
33. Kjeldsen K, Norgaard A, Clausen T, 1984. The age-dependent changes in the number of 3H-ouabain binding sites in mammalian skeletal muscles. Pflugers Arch. 402:100-108. [Medline]
34. Kjeldsen K, Gotzsche CO, Norgaard A, Thomassen A, Clausen T, 1984. Effect of thyroid function on number of Na+/K+ pumps in human skeletal muscle. Lancet. 2:8-10. [Medline]
35. Kjeldsen K, Richter EA, Henrik G, Lortie G, Clausen T, 1986. Training increases the number of [3H] ouabain-binding sites in rat skeletal muscle. Biochim Biophys Acta. 860:708-712. [Medline]
36. Mooradian AD, Wong NC, Shah GN, 1996. Age-related changes in the responsiveness of apolipoprotein A1 to thyroid hormone. Am J Physiol. 271:R1602-R1607. [Abstract/Free Full Text]
37. Mooradian AD, Fox-Robichaud A, Meijer ME, Wong NC, 1994. Relationship between transcription factors and S14 gene expression in response to thyroid hormone and age. Proc Soc Exp Biol Med. 207:97-101. [Abstract]
38. Mooradian AD, Scarpace PJ, 1993. 3,5,3'-L-triiodothyronine regulation of beta-adrenergic receptor density and adenylyl cyclase activity in synaptosomal membranes of aged rats. Neurosci Lett. 161:101-104. [Medline]
39. Clausen T, 1990. Regulation of active Na+-K+ transport in skeletal muscle. Physiol Rev. 66:542-590.
40. Everts ME, Retterstol K, Clausen T, 1988. Effects of adrenaline on excitation-induced stimulation of the sodium-potassium pump in skeletal muscle. Acta Physiol Scand. 134:189-198. [Medline]
41. Bianchi CP, Narayan S, 1981. Possible role of the transverse tubules in accumulating calcium released from the terminal cisternae by stimulation and drugs. Can J Physiol. 60:503-507.
42. Godt RE, Nosek TM, 1989. Changes of extracellular milieu with fatigue or hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle. J Physiol. 412:155-180. [Abstract/Free Full Text]

This article has been cited by other articles:

				K. F. Herspring, L. F. Ferreira, S. W. Copp, B. S. Snyder, D. C. Poole, and T. I. Musch Effects of antioxidants on contracting spinotrapezius muscle microvascular oxygenation and blood flow in aged rats J Appl Physiol, December 1, 2008; 105(6): 1889 - 1896. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Norton, M. W. Articles by McCarter, R. J.M. Search for Related Content PubMed PubMed Citation Articles by Norton, M. W. Articles by McCarter, R. J.M.