Effects of Aging on Diaphragm Contractile Function in Golden Hamsters1- 3
YILI ZHANG and STEVEN G. KELSEN
Introduction
Maximal static inspiratory and expiratory pressure is diminished with aging in normal adult human subjects (1). However, changes in muscle performance during voluntary contractions may reflect alterations in the function of the cardiovascular or somatic motor system as well as deterioration in the contractility of the respiratory muscles themselves (2-5). Study of the contractile performance of isolated electrically stimulated muscle bundles has allowed the effects of aging on the intrinsic contractile properties of limb muscles to be assessed independent of cardiovascular or central nervous system function. The present study, therefore, examined the effects of aging on the force-generating and shortening properties of the adult diaphragm using strips of the costal diaphragm stimulated elec·trically in vitro. Experiments were performed in the hamster, a species previously used to assess the effects of chronic alterations in respiratory mechanics (6) and nutritional state on respiratory muscle structure and function (7). Methods Studies were performed on 62 muscle strips from 42 Golden hamsters. Three groups of animals 4 to 5 (n = 15), 12 to 14 (n = 13), and 17 to 19 (n = 14) months of age at the time they were killed were studied. (The life span of the Golden hamster is approximately 22 months with 50070 of animals dying between 20 and 22 months [8]). Elderly animals were studied at 17 to 19 months of age rather than at 22 to 24 months of age to avoid possible bias related to "survivor" effects (4). The hamsters were killed by decapitation, and the entire diaphragm was removed and immersed in oxygenated Krebs-Henseleit solution within 5 min. Muscle strips (3 to 4 mm wide) were dissected from the costal region of the diaphragm as previously described (6, 7). The origin of the muscle with bone intact was mounted in a tissue bath by a clip. The central tendinous insertion was tied with 6.0 surgical thread to the torque arm of an ergometer (Model 300H; Cambridge Instruments, Hollis, NH). The Krebs solution in the 1396
SUMMARY The present study investigated the effects of aging on the contractile properties of the adult diaphragm in 42 Golden Syrian hamsters. Experiments were performed on isolated diaphragm strips from three groups of animals 4.9 ± 0.4 (SE), 12.8 ± 0.2, and 18.8 ± 0.3 months of age. Aging was associated with a decrease in the maximal Isometric tension generated per unit cross-sectional area of muscle and slowing In the time-to-peak tension and rate of muscle relaxation. The velocity of muscle shortening at a given load was also significantly less in older than in younger animals, and the force-velocity curve became flatter with advancing age as reflected by Increases in the Hili coefficient (alP.). Changes in maximal active tension and maximal velocity of unloaded shortening with aging correlated weakly (r >0.3; p < 0.05) and were of similar magnitude (-17 to -21%), suggesting that aging affects these two Indices of muscle function In similar fash· Ion. Finally, the diaphragm fatigued more rapidly in older than In younger animals. We conclude that aging depresses the ability of the adult diaphragm to generate tension and to shorten and AM REV RESPIR DIS 1990; 142:1396-1401 resist fatigue.
tissue bath was oxygenated with a 95% O 2-5% CO 2 gas mixture and maintained at a temperature of 24.5 ± 0.5 0 C and a pH of 7.40 ± 0.05. Composition of the aerated Krebs-Henseleit solution in mEq/L was as follows: Na+, 153.8; K+, 5.0; Ca 2+, 5.0; Mg2+, 2.0; Cl-, 145.0; HC0 3 -, 15.0; HPO/-, 1.9; SO/-, 2.0; glucose, 110 mg%; regular crystalline zinc insulin, 50 U. Strips were stimulated electrically (Model S88 stimulator and constant current isolation unit; Grass Instruments, Quincy, MA) with 0.2-ms biphasic impulses delivered via two platinum electrodes placed on both sides of the muscle. Supramaximal stimulation (Le., 1.2 to 1.5 times the current required to elicit maximal twitch tension, 6 to 12 V) was used throughout the experiment. A dual mode servo ergometer system (Model 300H; Cambridge Technology, Inc.) was used to measure and control muscle load and displacement during both isometric and isotonic contractions. In the isometric mode of operation, the system has a step response time for force of 100% of 1.0 ms. The compliance of the torque arm is 0.3 Ilm/g. In the isotonic mode, the force resolution is less than 0.020 g, and the accuracy of displacement is 2%. A controller circuit, composed of several multivibrators (MCI4538B) and several operational amplifiers (MCI741), was used to generate step changes in force or displacement and deliver stimulus pulses at preset times during individual isotonic and isometric contractions. The muscle bath was mounted on a movable chassis whose position was altered using a micrometer-controlled rack and pinion gear (accuracy of displacement, 0.005 mm). Mus-
cle fiber length was altered by moving the position of the bath and measured with a micrometer in close proximity to the muscle. The optimal length of the muscle (La) was defined as the fiber length at which maximal active twitch tension (0.2-ms pulses delivered at 1 Hz) was generated. Muscle fiber length was measured with microcalibers. The response of the muscle to increasing stimulus frequency (Le., the force-frequency relationship) was assessed at La by applying 10, 20, 25, 30,40, 50, 60, 80, 100, and 150 Hz pulses applied in I-s trains. A 2-min period elapsed between contractions. The active length-tension relationship of the muscle was assessed from peak tetanic tension at 75,80,85,90, 100, and 115% La. Tetanic tension was measured in response to 50 Hz electrical stimuli applied for 1 s. The isotonic force-velocity relationship was determined by the quick release method while the muscle was tetanized (50 Hz stimuli). At
(Received in originalform September 22, 1989 and in revised form May 7, 1990) 1 From the Pulmonary Division, Department of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania. 2 Supported in part by Grant No. HL-40295-01 from the National Institutes of Health. 3 Correspondence and requests for reprints should be addressed to Steven G. Keisen, M.D., Department of Medicine, Pulmonary Division, Temple University Hospital, Room 931, Parkinson Pavilion, Broad and Tioga Streets, Philadelphia, PA 19140.
AGING AND THE DIAPHRAGM
1397
o
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Fig. 1. Relationship of isotonic velocity of shortening to externai load (expressed as PIP.) in a single muscle strip during quick-release afterloaded contractions.
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0.25 s after the onset of an isometric contraction (by which time tension plateaued), a step decrease in load was introduced to the torque arm of the ergometer allowing the muscle to shorten isotonically. The procedure was repeated at 7 to 10 different levels of afterload. Velocity of shortening was calculated from changes in displacement, which occurred 10 to 30 ms after the release during which displacement is linear with time. The velocity of shortening on a given load was obtained from the slope of the straight line relating displacement against time using the least-squares regression method. The force-velocity relationship over a range of loads from 1 to 80070 maximal tension was assessed using a modification of Hill's equation (9): V (P + a)
=
(Po - P)b
(1)
where - bP 0 + aV + bP + VP = 0
(2)
(V = velocity on a given load; P = tension at a given load; Po = maximal isometric tension.) Correlation coefficients for the relationship between tension and velocity exceeded 0.92 in all trials (figure 1). The velocity of shortening at a given level of load (Le., 0.1, 0.2, 0.3, and 0.4 P/P o) was determined in each muscle strip by interpolation of the forcevelocity relationship. The extrapolated unloaded shortening velocity during quickrelease experiments (Voq) was calculated as: Voq = bPo/a, and the coefficients a and b were computed by least-squares regression in each muscle strip. The slack test method of Edman (10) was also used to assess the maximal velocity (Vmax) of unloaded shortening. After isometric tension reached a plateau, an abrupt step change in the position of the torque arm was introduced to reduce muscle fiber length. The introduction of slack caused tension to fall rapidly to zero « 1 ms). Thereafter, tension was zero until the muscle had shortened sufficiently to take up the slack. The ratio of the change in muscle length over the time needed to take up slack and reapply tension
was used to measure the velocity of unloaded shortening. Several (three to four) amplitudes of torque arm displacement were used in each strip. The intervals during which tension was zero (AT) were related to the step changes in length (AL) by least-squares regression. Maximal velocity of shortening during the slack test (Vos) was calculated as the slope of the line relating AL to AT. Correlation coefficients for the relationship between AL versus AT exceeded 0.95 in all experiments (figure 2). To determine whether 50 Hz stimuli achieved maximal shortening responses, in eight strips, the slack test was performed in separate trials at 150 and 50 Hz, and the results were compared. To assess muscle endurance, rhythmic isometric contractions were performed for a 5-min period, and the rate of fall in peak isometric tension was measured. (Stimulus frequency was 20 Hz, contraction rate was 90 min, and duty cycle, Le., the ratio of contraction time [0.22 s] over total cycle time [0.66 s] was 0.33.) Muscle endurance was assessed from the rate of fall of peak isometric tension. Total lung capacity was measured at the time of death as previously described (6). Briefly, the trachea was cannulated, and the lung was inflated with saline to an airway pressure of 30 cm H 2 0. Lung volume was measured by water displacement.
Data Analysis A Minc 11/23 computer was used for on-line data acquisition and analysis. The sampling
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Time (msec) Fig. 2. Relationship of displacement (~L) and time during which tension was zero (~T) in slack test performed in a single muscle strip.
frequencies in quick-release and slack-test experiments were 200 and 5,000 Hz, respectively. Statistical significance of differences in group mean data for different age groups was examined by ANOVA and by independent t test. Results
Age, body weight, and lung volume of the three groups of animals are shown in table 1. As may be seen, total lung capacity increased significantly with advancing age, but there was no statistically significant difference in body weight in the three groups. Increasing age affected the forcefrequency relationship (figure 3). The force-frequency curve (where force is expressed as a percentage of maximum) obtained in elderly animals was displaced to the left of that obtained in young animals (p = 0.0025 for the entire curve). That is, over the range of frequencies from 10 to 40 Hz, diaphragms from older animals generated a greater percent-
TABLE 1 CHARACTERISTICS OF ANIMALS'
Group Young adult Middle-aged Elderly
Age (months)
Body Weight (g)
Total Lung Volume (m/)
4.9 ± 0.4 12.8 ± 0.2 18.8 ± 0.3
161.9 ± 5.2 165.0 ± 5.1 162.6 ± 8.3
6.0 ± 0.3 7.5 ± O.4t 7.7 ± 0.3t
* Values are mean ± SE.
t
p
< 0.02 for comparison with young adult group.
1398
ZHANG AND KELSEN
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Fig. 3. Force-frequency relationship in young adult (crosses) and elderly (triangles) animals. The relationship is displaced to the left in elderly relative to young adult animals (p = 0.0025).
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age of maximal tension than did diaphragms from younger animals (p < 0.0002 by ANOVA). From 50 to 150 Hz, however, there was no significant difference in the curves obtained in the two groups of animals (p > 0.50). Moreover, there was no significant difference in tension generated at 50 Hz between old and young animals (p > 0.10 by t test). Increasing age was, however, associated with a decrease in maximal isometric tension expressed in absolute terms (kg/cm2 ) (p < 0.001 by ANOVA) and a reduction in tension at all lengths (p < 0.0001 by ANOVA) (figure 4). The fiber length at which active tension was maximal was unaffected by aging, however (p > 0.1 by t test). Maximal tension at L o was significantly less in muscles from elderly (Le., 18.8 months old) than from young adult (i.e., 4.8 months old) and middle-aged (12.8 months old) animals. However, maximal tension was not significantly different in younger and middle-aged animals (p > 0.25). The twitchltetanus ratio increased significantly with aging (p < 0.001 by ANOVA) and was greater in the older group than in the two younger groups (p < 0.02 by t test for both comparisons). Aging was also associated with a slowing in the twitch tension wave form. Contraction time (CT) and one-half relaxa-
tion time (112 RT) were significantly prolonged (p < 0.0001 by ANOVA for CT and Y2RT) and were greater in the elderly group than in the other two groups (p < 0.02 for both comparisons). There was no difference in CT or Y2RT in diaphragms from the two younger groups of animals, however (p > 0.25 by t test) (table 2). Aging also altered the force-velocity relationship of the diaphragm (figure 5). The velocity of shortening at a given submaximal load (Le., P/P o) decreased with aging (p < 0.0001 by ANOVA for the whole force-velocity curve). Furthermore, the constants a, b, and alP 0 of the Hill equation (table 3) were significantly greater in the older than in the younger animals (p < 0.001 by ANOVA for all comparisons). The maximal velocity of shortening assessed by the slack test (Le., Vos) and by extrapolation of the isotonic force-velocity relationship (Le., Voq) also decreased with aging (p < 0.0001 by ANOVA for both Vos and Voq) (figure 6). Of note, Vos was similar during 50 and 150 Hz stimuli (e.g., 6.25 ± 0.79 SE Lois and 6.44 ± 0.57 SE Lois, respectively; p = 0.69). Maximal velocity of shortening in the entire population of animals correlated weakly with maximal tetanic tension (kg/cm 2 ) at L o (r = 0.32 and 0.41 for Vos
TABLE 2 EFFECT OF AGING ON MAXIMAL TENSION AND THE TWITCH WAVE FORM*
Group Young adult Middle-aged Elderly
Maximal Tension (kg/cm 2 )
L. (mm)
CT (ms)
'hRT (ms)
TwitchiTetanus
2.10 ± 0.10 2.05 ± 0.07 1.70 ± 0.04*
13.8 ± 0.45 14.3 ± 0.31 13.4 ± 0.35
58.60 ± 1.44 59.73 ± 2.13 72.62 ± 1.91 t
54.20 ± 2.23 57.87 ± 2.09 75.54 ± 3.82t
0.49 ± 0.02 0.49 ± 0.01 0.54 ± O.Olt
Definition of abbreviations: L, = optimal length of muscle; CT = contraction time; 'hRT • Values are mean ± SE. t p < 0.02 for comparison with young adult and middle-aged groups.
= one-half
relaxation time.
,
,
0.9 1.1 FIBER LENGTH (L/L ) o
,
1.3
Fig. 4. Length-active tension relationship of the hamster diaphram in young adult (crosses), middle-aged (circles), and elderly (triangles) animals. Symbols represent mean ± SE. Maximal tension at L. is significantly less in muscles from the elderly group than in those from the two younger groups (p < 0.05 for both), but it is not significantly different in animals in the two younger groups (p < 0.25). The fiber length at which active tension is maximum is similar in all three groups (p < 0.1).
and Voq against tetanic tension, respectively; p < 0.05 for both). The Hill coefficient, alP 0, also correlated significantly with maximal tetanic tension (r = 0.49; p < 0.005). Reductions in maximal velocity (Vmax) were also correlated with prolongations in CT and 112 RT (r = - 0.62 and - 0.49 for the relationship of Vos versus CT and Y2RT, respectively, and r = -0.45 and -0.38 for the relationship ofVoq versus CT and Y2RT, respectively; p < 0.01 for all comparisons). Aging also was associated with a decrease in diaphragmatic muscle endurance (p < 0.002 by ANOVA) (figure 7). Tension fell more rapidly in diaphragms from elderly animals than in muscles from animals in the two younger groups, and it was significantly less at the end of the fatigue trial (i.e., at 5 min) in the elderly group when compared with that in the two younger groups (p < 0.01 by t test for both). Discussion
The present study indicates that aging in Golden hamsters is associated with alterations in the contractile properties in the diaphragm. The maximal tensiongenerating ability of the diaphragm is diminished; twitch tension development and relaxation become slower; the forcefrequency relationship is displaced to the left; the force-velocity curve is displaced to the left and assumes a less concave shape; maximal shortening velocity decreases; and the endurance of the di-
1399
AGING AND THE DIAPHRAGM
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Fig. 6. Velocity of shortening at zero load ("max) measured by the slack test method (Vos) (open bar) and by extrapolation of Hill's equation (Voq) (hatched bar) in young, middle-aged, and elderly animals. Symbols represent mean ± SE. "max decreases with aging (p < 0,01 for Vos and p < 0.025 for Voq) and is significantly reduced in the elderly compared with that in the two younger groups (p < 0,01 for both methods).
I
M
AGE
M
FORCE(~P~ Fig. 5. Isotonic force-velocity relationship of the hamster diaphragm in young adult (crosses), middle-aged (circles), and elderly (triangles) animals. Symbols represent mean ± SE. The relationship is less steep (Le., alP. is greater) in the older than in the younger animals. Furthermore, both the extrapolated "max and velocity at submaximalloads are significantly reduced in elder.Iy compared with young adult and middle-aged animals (p < 0,01 for all comparisons).
aphragm in response to subtetanic stimuli is diminished. Interestingly, changes in diaphragm function did not appear to be progressive throughout adulthood. Rather, changes occurred only in the oldest group (i.e., 18.8 months) and were not apparent in 4.9- and 12.8-month-old groups. It is important to point out in this regard that the present study was performed in hamsters over a wide range of the adult life span but before extreme senescence to avoid "survivor" effects caused by attrition in the elderly group. Of note, values for maximal tetanic tension (kg/cm2 ), CT, and YzRT, and extrapolated Vmax obtained in the hamster diaphragm in 4.9- and 12.8-monthold animals resemble those obtained by Metzger and coworkers (11) and Faulkner
Interpretation of Results Displacement of the force-frequency relationship in elderly animals appears to be attributable to prolongation in CT and Yz RT, with greater propensity to develop a fused contraction. Alterations in the force-frequency relationship caused diaphragms from elderly animals to generate a greater percentage of maximal tension at stimulus frequencies of 40 Hz and below, and this is likely to have contributed to the greater rate of fall in diaphragm tension during fatigue trials in these older animals. Isometric tension in response to the 20-Hz stimulus was 89 ± 20/0 SE and 84 ± 2% SEofmaximuminelderly and young animals, respectively (p < 0.05), tending at a given duty cycle (i.e., 0.33) to produce a greater tension-time index during the fatigue trial in muscles from elderly animals (13). Changes in maximal isometric tension and velocity of shortening do not appear to be explained by differences in the
TABLE 3
Correlation Coefficients
Young adult Middle-aged Elderly
0'
o
1
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~~-t-
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2
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3
1
4
I
5
TIME (min) Fig. 7. Effect of age on the rate of decline in peak diaphragm tension during repeated contraction in young adult (crosses), middle-aged (circles), and elderly (triangles) animals. Symbols represent mean ± SE. Tension fell more rapidly in diaphragms from elderly animals than in muscles from young adult and middle-aged animals (p < 0.002) and is significantly less at the end of the fatigue trial (Le., 5-min point) in the elderly group than in the two younger groups (p < 0,01 for both).
and colleagues (12) in the rat diaphragm in young adult animals.
EFFECT OF AGING ON THE VELOCITY OF DIAPHRAGM SHORTENING"
Group
"
")~~-a:
o
>
a (g)
b (mm/s)
alP.
6.12 ± 0.56 7.26 ± 0.45 12.09 ± o.oat
6.96 ± o.oa 9.97 ± 0.45 13.11 ± 0.09t
0.16 ± 0.01 0.20 ± 0.02 0.34 ± 0.04t
Definition of abbreviations: Po = maximal isometric tension. " Values are mean ± SE. t p < 0.001 for comparison of all these groups.
force-frequency relationship with aging, however. Tension generated in response to 50 Hz stimuli was 1000/0 of maximum in both young adult and elderly animals. Moreover, maximal velocity of shortening in response to 50 and 150 Hz stimuli was similar, indicating that 50 Hz stimuli achieved full activation during shortening maneuvers in muscles from young and from old animals. In limb muscle, reductions in contractile protein content appear to explain the decrease in maximal tension (14-21). When tension is corrected for alterations in muscle cross-sectional area, however, maximal tension is generally unchanged with aging (14-19), but it may be reduced (20). Although reductions in maximal diaphragmatic tension per unit cross-sectional area in elderly hamsters could represent decreases in the tension-generating ability of the residual myofibrillar material, reductions in maximal tension per unit cross-sectional area of diaphragmatic muscle may simply reflect the presence of noncontractile material that does not contribute to tension development. Detailed examination of muscle structure is needed to test the second possibility. Of note, the shape of the length-tension curve and fiber length at L o were unaffected by aging, which is in agreement with previous results by McCarter and McGee (19) and Brooks and Faulkner (20). The present study also demonstrated that aging is associated with alterations in the force-velocity relationship of the
ZHANG AND KELSEN
1400
diaphragm and the curvature of the forcevelocity relationship, as indicated by the Hill constant, alP o. Reductions in the velocity of diaphragmatic muscle shortening, increases in alP o, and prolongations in CT and Y2RT make the aged diaphragm more closely resemble a slow muscle (22). Changes observed may, therefore, represent alterations in muscle fiber composition in elderly animals. In limb skeletal muscle, aging is associated with a preferential atrophy of fast twitch fibers with the result that slow fibers make up a greater percentage of muscle mass (16,17). There is little information about the effects of aging on fiber composition of the respiratory muscles. We have observed that the cross-sectional area of fast twitch muscle fibers in the intercostal muscles is smaller in 18-month-old hamsters, and a greater percentage of the muscle mass is composed of slow twitch muscle fibers compared with the muscle mass in 12month-old animals (23). The changes in fast twitch fiber size in the intercostal muscles, therefore, appear to be similar to what has been observed in limb muscle. Changes in fiber composition, however, do not appear to be the sole explanation for the effects of aging on diaphragmatic muscle function for several reasons. First, the maximal velocity of unloaded shortening assessed by the slack test is decreased. Values obtained in the slack test appear to be determined by the rate of shortening of the most rapidly shortening fibers, Le., fast twitch fibers (24), suggesting that the velocity of shortening of the fast fiber population is diminished. Second, recent studies indicate that the contraction and relaxation time of individual fast twitch and slow twitch motor units in the soleus and extensor digitorum longus muscles increase with aging (25). Reductions in the speed of shortening and tension kinetics of diaphragm muscle bundles, therefore, may be a reflection of changes in the properties of individual fast and slow motor units. Third, the reduction in the endurance of the diaphragm in elderly animals is not explainable by preferential fast fiber atrophy since such a change in fiber composition should have increased endurance.
The Effect of Aging on Limb Skeletal Muscle A large body of literature obtained from studies on limb skeletal muscles in a variety of species indicates that aging is associated with reductions in muscle
weight, fiber diameter and fiber number, myofibrillar protein content, and the ability to synthesize high-energy phosphates (1,2,3, 16, 19,20,25). Aging also appears to be associated with subtle changes in the structure and function of the motor unit and myoneural junction (3,4). Although it is quite clear that aging per se influences the structure and biochemical properties of limb skeletal muscle, recent studies indicate that there are surprisingly few changes in maximal tension, shortening velocity, and endurance of limb-skeletal muscles excited electrically in vitro (14, 15, 19). For example, maximal active tension (Le., kg/cm 2 ) of the soleus, a slow muscle (14, 15, 19), the extensor digitorum longus (EDL), a fast muscle (14, 15), and the omohyoideus, a mixed muscle (19), is similar in young adult (Le., 9 months old) and elderly (Le., 28 to 30 months old) rats. In fact, Eddinger and coworkers (14) found that maximal tension per unit of cross-sectional area in single fibers of the soleus was greater in 30-month-old than in 9-month-old rats. On the other hand, Brooks and Faulkner (20) noted a 22010 reduction in maximal tension in whole muscle bundles of the EDL in elderly mice. (Of note, maximal soleus tension was unaffected by aging [20].) Similarly, there appears to be no reduction in Vmax determined either by the slack test (14) or by extrapolation of the isotonic force-velocity relationship (14, 15, 20) in the soleus or the EDL (20). In fact, Eddinger and coworkers (14) observed an increase in Vmax determined by the slack test in soleus muscle bundles and skinned single fibers. On the other hand, extrapolated Vmax and isotonic velocity of shortening at submaximal loads (Le., PI Po 0.1 to 0.9) of the EDL was reduced in 30-month-old compared with 9-month-old animals (14). Finally, several studies have observed no effect of aging on the endurance of the soleus (14, 19), EDL (14), or omohyoideus (19) muscles during electrically stimulated contractions in vitro. The above results on the effects of aging on maximal tension, shortening velocity, and endurance in limb skeletal muscles are in contrast to the results we obtained in the diaphragm where large reductions in performance were observed. Respiratory skeletal muscle, like skeletal muscle in other parts of the body, is highly plastic and alters its structure, Qiochemical properties, and contractile function in response to alterations in applied load, precontraction length, and
pattern of activity (6, 27). Aging is associated with changes in the mechanics of breathing, lung gas exchange, and chemosensitivity to hypoxia and hypercapnia, which may alter the load and level of activity of the diaphragm (28-30). Moreover, the activity level of older animals may be less than that of younger animals. Reductions in the level of ventilation in older compared with younger animals (as a result of reductions in physical activity or reductions in respiratory motor activity at a given level of physical activity) could have contributed to the changes in muscle function observed. However, we know of no data in which respiratory motor activity has been compared in young and old animals. Regardless of mechanism(s), the results of the present study are likely to apply to humans. For example, decreases in maximal diaphragmatic tension with aging in the hamster appear to have a similar time course and magnitude to reductions in inspiratory muscle strength assessed from maximal static inspiratory pressure in normal human subjects (1). Maximal static inspiratory pressure in elderly (Le., 80-yr-old) male and female subjects is approximately 30% lower than in young (i.e., 20- to 30-year-old) male and female subjects and does not appear to decrease significantly with aging until after approximately 50 to 60 yr of age (reference 1 and figures 2 and 3). References 1. Black LF, Hyatt RE. Maximal respiratory pressures: normal values and relationship to age and sex. Am Rev Respir Dis 1968; 99:696-702. 2. Larsson L, arimby 0, Karlsson J. Muscle strength and speed of movement in relation to age and muscle morphology. J Appl Physiol 1979; 451-6. 3. Brown WE A method for estimating the number of motor units in thenar muscles and the changes in motor unit count with aging. J Neurol Neurosurg Psychiatry 1972; 35:845-52. 4. Banker BQ, Kelly SS, Robbins, N. Neuromuscular transmission and correlative morphology in young and old mice. J Physiol (Lond) 1983; 339: 355-75. 5. Chesky JA. Comparative aspects of declining muscle function. Aging 1978; 6:179-200. 6. Supinski as, Kelsen so. Effect of elastaseinduced emphysema on the force generating ability of diaphragm. J Clin Invest 1982; 70:978-88. 7. Kelsen SO, Ference M, Kapoor S. Effects of prolonged undernutrition on structure and function of the diaphragm. J Appl Physiol 1985; 58:1354-9. 8. Orsini MW. Inbred and genetically defined strains of laboratory animals. Part 2. Hamster, guinea pig, rabbit and chicken. In: Altman PL, Katz DD, eds. Biological handbooks III. Federation of American Societies for Experimental Biology, 1979; 436-7. 9. Hill AV. The heat of shortening and dynamic
AGING AND THE DIAPHRAGM
constants of muscle. Proc R Soc London [Bioi] 1938; 126:136-95. 10. Edman KAP. The velocity of unloaded shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibers. J Physiol (Lond) 1979; 291:143-59. 11. Metzger JM, Schmidt KB, Fitts RH. Histochemical and physiological characteristics of the rat diaphragm. J Appl Physiol 1985; 58:1085-91. 12. Faulkner JA, Claflin DR, McCullyKK, Jones AD. Contractile properties of bundles of fiber segments from skeletal muscles. Am J Physiol 1982; 243:C66-73. 13. Kelsen SG, Nochomovitz ML. Fatigue of the mammalian diaphragm in vitro. J Appl Physiol 1982; 53:440-7. 14. Eddinger n, Cassens RG, Moss RL. Mechanical and histochemical characterization of skeletal muscles from senescent rats. Am J Physiol 1986; 251:C421-30. 15. Fitts RH, Troup JP, Witzmann FA, Holloszy JO. The effect of aging and exercise on skeletal muscle function. Mech Ageing Dev 1984; 27:161-72. 16. Gutmann E, Hanzlikova V, Vyskocil F. Age changes in cross striated muscle of the rat. J Phys-
1401 iol (Lond) 1971; 219:331-43. 17. Gutmann E, Syrovy I. Contraction properties and myosin ATPase activity of fast and slow senile muscles of the rat. Gerontology 1974; 20:239-44. 18. Larsson 1. Aging in mammalian skeletal muscle. In: Mortimer JA, Pirozzolo FJ, Maletta GJ, eds. The aging motor system. New York: Praeger, 1982; 61-97. 19. McCarter R, McGee MRM. Influence of nutrition and aging on the composition and function of rat skeletal muscle. J Gerontol1987; 42:432-41. 20. Brooks SV, Faulkner JA. Contractile properties of skeletal muscles from young, adult and aged mice. J Physiol (Lond) 1988; 404:71-82. 21. Daw CK, Starnes JW, White TP. Muscle atrophy and hypoplasia with aging: impact of training and food restriction. J Appl Physiol 1988; 64: 2428-32. 22. Ranatunga KW. The force-velocity relation of rat fast- and slow-twitch muscles examined at different temperatures. J Physiol (Lond) 1984; 351:517-29. 23. Bao S, Thomas AJ, Kelsen SG. Structure of intercostal muscle: topographic and age-dependent effects (abstract). Fed Proc 1987; 43:818. 24. Dennis RC, Faulkner JA. Shortening veloci-
ty extrapolated to zero load and unloaded shortening velocity of whole rat skeletal muscle. J Physiol (Lond) 1985; 359:357-63. 25. Edstrom L, Larson 1. Effects of age on contractile and enzyme-histochemical properties of fastand slow-twitch single motor units in the rat. J Physiol (Lond) 1987; 392:129-45. 26. Moller P, Bergstron J, Furst P, Hellstrom K. Effect of aging on energy-rich phosphagen in human skeletal muscles. Clin Sci 1980; 58:553-5. 27. Jolesz F, Sreter FA. Development, innervation and activity-pattern induced changes in skeletal muscle. Annu Rev Physiol 1981; 43:531-52. 28. Kronenberg RS, Drage CWo Attenuation of the ventilatory and heart rate responses to hypoxia and hypercapnia with aging in normal men. J Clin Invest 1973; 52:1812-9. 29. Holland J, Milic-Emili J, Macklem PT, Bates DV. Regional distribution of pulmonary ventilation and perfusion in elderly subjects. J Clin Invest 1968; 47:81-92. 30. Turner JM, Mead J, Wohl ME. Elasticity of human lungs in relation to age. J Appl Physiol1968; 25:664-71.
YILI ZHANG and STEVEN G. KELSEN
Introduction
Maximal static inspiratory and expiratory pressure is diminished with aging in normal adult human subjects (1). However, changes in muscle performance during voluntary contractions may reflect alterations in the function of the cardiovascular or somatic motor system as well as deterioration in the contractility of the respiratory muscles themselves (2-5). Study of the contractile performance of isolated electrically stimulated muscle bundles has allowed the effects of aging on the intrinsic contractile properties of limb muscles to be assessed independent of cardiovascular or central nervous system function. The present study, therefore, examined the effects of aging on the force-generating and shortening properties of the adult diaphragm using strips of the costal diaphragm stimulated elec·trically in vitro. Experiments were performed in the hamster, a species previously used to assess the effects of chronic alterations in respiratory mechanics (6) and nutritional state on respiratory muscle structure and function (7). Methods Studies were performed on 62 muscle strips from 42 Golden hamsters. Three groups of animals 4 to 5 (n = 15), 12 to 14 (n = 13), and 17 to 19 (n = 14) months of age at the time they were killed were studied. (The life span of the Golden hamster is approximately 22 months with 50070 of animals dying between 20 and 22 months [8]). Elderly animals were studied at 17 to 19 months of age rather than at 22 to 24 months of age to avoid possible bias related to "survivor" effects (4). The hamsters were killed by decapitation, and the entire diaphragm was removed and immersed in oxygenated Krebs-Henseleit solution within 5 min. Muscle strips (3 to 4 mm wide) were dissected from the costal region of the diaphragm as previously described (6, 7). The origin of the muscle with bone intact was mounted in a tissue bath by a clip. The central tendinous insertion was tied with 6.0 surgical thread to the torque arm of an ergometer (Model 300H; Cambridge Instruments, Hollis, NH). The Krebs solution in the 1396
SUMMARY The present study investigated the effects of aging on the contractile properties of the adult diaphragm in 42 Golden Syrian hamsters. Experiments were performed on isolated diaphragm strips from three groups of animals 4.9 ± 0.4 (SE), 12.8 ± 0.2, and 18.8 ± 0.3 months of age. Aging was associated with a decrease in the maximal Isometric tension generated per unit cross-sectional area of muscle and slowing In the time-to-peak tension and rate of muscle relaxation. The velocity of muscle shortening at a given load was also significantly less in older than in younger animals, and the force-velocity curve became flatter with advancing age as reflected by Increases in the Hili coefficient (alP.). Changes in maximal active tension and maximal velocity of unloaded shortening with aging correlated weakly (r >0.3; p < 0.05) and were of similar magnitude (-17 to -21%), suggesting that aging affects these two Indices of muscle function In similar fash· Ion. Finally, the diaphragm fatigued more rapidly in older than In younger animals. We conclude that aging depresses the ability of the adult diaphragm to generate tension and to shorten and AM REV RESPIR DIS 1990; 142:1396-1401 resist fatigue.
tissue bath was oxygenated with a 95% O 2-5% CO 2 gas mixture and maintained at a temperature of 24.5 ± 0.5 0 C and a pH of 7.40 ± 0.05. Composition of the aerated Krebs-Henseleit solution in mEq/L was as follows: Na+, 153.8; K+, 5.0; Ca 2+, 5.0; Mg2+, 2.0; Cl-, 145.0; HC0 3 -, 15.0; HPO/-, 1.9; SO/-, 2.0; glucose, 110 mg%; regular crystalline zinc insulin, 50 U. Strips were stimulated electrically (Model S88 stimulator and constant current isolation unit; Grass Instruments, Quincy, MA) with 0.2-ms biphasic impulses delivered via two platinum electrodes placed on both sides of the muscle. Supramaximal stimulation (Le., 1.2 to 1.5 times the current required to elicit maximal twitch tension, 6 to 12 V) was used throughout the experiment. A dual mode servo ergometer system (Model 300H; Cambridge Technology, Inc.) was used to measure and control muscle load and displacement during both isometric and isotonic contractions. In the isometric mode of operation, the system has a step response time for force of 100% of 1.0 ms. The compliance of the torque arm is 0.3 Ilm/g. In the isotonic mode, the force resolution is less than 0.020 g, and the accuracy of displacement is 2%. A controller circuit, composed of several multivibrators (MCI4538B) and several operational amplifiers (MCI741), was used to generate step changes in force or displacement and deliver stimulus pulses at preset times during individual isotonic and isometric contractions. The muscle bath was mounted on a movable chassis whose position was altered using a micrometer-controlled rack and pinion gear (accuracy of displacement, 0.005 mm). Mus-
cle fiber length was altered by moving the position of the bath and measured with a micrometer in close proximity to the muscle. The optimal length of the muscle (La) was defined as the fiber length at which maximal active twitch tension (0.2-ms pulses delivered at 1 Hz) was generated. Muscle fiber length was measured with microcalibers. The response of the muscle to increasing stimulus frequency (Le., the force-frequency relationship) was assessed at La by applying 10, 20, 25, 30,40, 50, 60, 80, 100, and 150 Hz pulses applied in I-s trains. A 2-min period elapsed between contractions. The active length-tension relationship of the muscle was assessed from peak tetanic tension at 75,80,85,90, 100, and 115% La. Tetanic tension was measured in response to 50 Hz electrical stimuli applied for 1 s. The isotonic force-velocity relationship was determined by the quick release method while the muscle was tetanized (50 Hz stimuli). At
(Received in originalform September 22, 1989 and in revised form May 7, 1990) 1 From the Pulmonary Division, Department of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania. 2 Supported in part by Grant No. HL-40295-01 from the National Institutes of Health. 3 Correspondence and requests for reprints should be addressed to Steven G. Keisen, M.D., Department of Medicine, Pulmonary Division, Temple University Hospital, Room 931, Parkinson Pavilion, Broad and Tioga Streets, Philadelphia, PA 19140.
AGING AND THE DIAPHRAGM
1397
o
5
6
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Fig. 1. Relationship of isotonic velocity of shortening to externai load (expressed as PIP.) in a single muscle strip during quick-release afterloaded contractions.
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0.25 s after the onset of an isometric contraction (by which time tension plateaued), a step decrease in load was introduced to the torque arm of the ergometer allowing the muscle to shorten isotonically. The procedure was repeated at 7 to 10 different levels of afterload. Velocity of shortening was calculated from changes in displacement, which occurred 10 to 30 ms after the release during which displacement is linear with time. The velocity of shortening on a given load was obtained from the slope of the straight line relating displacement against time using the least-squares regression method. The force-velocity relationship over a range of loads from 1 to 80070 maximal tension was assessed using a modification of Hill's equation (9): V (P + a)
=
(Po - P)b
(1)
where - bP 0 + aV + bP + VP = 0
(2)
(V = velocity on a given load; P = tension at a given load; Po = maximal isometric tension.) Correlation coefficients for the relationship between tension and velocity exceeded 0.92 in all trials (figure 1). The velocity of shortening at a given level of load (Le., 0.1, 0.2, 0.3, and 0.4 P/P o) was determined in each muscle strip by interpolation of the forcevelocity relationship. The extrapolated unloaded shortening velocity during quickrelease experiments (Voq) was calculated as: Voq = bPo/a, and the coefficients a and b were computed by least-squares regression in each muscle strip. The slack test method of Edman (10) was also used to assess the maximal velocity (Vmax) of unloaded shortening. After isometric tension reached a plateau, an abrupt step change in the position of the torque arm was introduced to reduce muscle fiber length. The introduction of slack caused tension to fall rapidly to zero « 1 ms). Thereafter, tension was zero until the muscle had shortened sufficiently to take up the slack. The ratio of the change in muscle length over the time needed to take up slack and reapply tension
was used to measure the velocity of unloaded shortening. Several (three to four) amplitudes of torque arm displacement were used in each strip. The intervals during which tension was zero (AT) were related to the step changes in length (AL) by least-squares regression. Maximal velocity of shortening during the slack test (Vos) was calculated as the slope of the line relating AL to AT. Correlation coefficients for the relationship between AL versus AT exceeded 0.95 in all experiments (figure 2). To determine whether 50 Hz stimuli achieved maximal shortening responses, in eight strips, the slack test was performed in separate trials at 150 and 50 Hz, and the results were compared. To assess muscle endurance, rhythmic isometric contractions were performed for a 5-min period, and the rate of fall in peak isometric tension was measured. (Stimulus frequency was 20 Hz, contraction rate was 90 min, and duty cycle, Le., the ratio of contraction time [0.22 s] over total cycle time [0.66 s] was 0.33.) Muscle endurance was assessed from the rate of fall of peak isometric tension. Total lung capacity was measured at the time of death as previously described (6). Briefly, the trachea was cannulated, and the lung was inflated with saline to an airway pressure of 30 cm H 2 0. Lung volume was measured by water displacement.
Data Analysis A Minc 11/23 computer was used for on-line data acquisition and analysis. The sampling
I
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10
20
30
40
Time (msec) Fig. 2. Relationship of displacement (~L) and time during which tension was zero (~T) in slack test performed in a single muscle strip.
frequencies in quick-release and slack-test experiments were 200 and 5,000 Hz, respectively. Statistical significance of differences in group mean data for different age groups was examined by ANOVA and by independent t test. Results
Age, body weight, and lung volume of the three groups of animals are shown in table 1. As may be seen, total lung capacity increased significantly with advancing age, but there was no statistically significant difference in body weight in the three groups. Increasing age affected the forcefrequency relationship (figure 3). The force-frequency curve (where force is expressed as a percentage of maximum) obtained in elderly animals was displaced to the left of that obtained in young animals (p = 0.0025 for the entire curve). That is, over the range of frequencies from 10 to 40 Hz, diaphragms from older animals generated a greater percent-
TABLE 1 CHARACTERISTICS OF ANIMALS'
Group Young adult Middle-aged Elderly
Age (months)
Body Weight (g)
Total Lung Volume (m/)
4.9 ± 0.4 12.8 ± 0.2 18.8 ± 0.3
161.9 ± 5.2 165.0 ± 5.1 162.6 ± 8.3
6.0 ± 0.3 7.5 ± O.4t 7.7 ± 0.3t
* Values are mean ± SE.
t
p
< 0.02 for comparison with young adult group.
1398
ZHANG AND KELSEN
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Fig. 3. Force-frequency relationship in young adult (crosses) and elderly (triangles) animals. The relationship is displaced to the left in elderly relative to young adult animals (p = 0.0025).
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age of maximal tension than did diaphragms from younger animals (p < 0.0002 by ANOVA). From 50 to 150 Hz, however, there was no significant difference in the curves obtained in the two groups of animals (p > 0.50). Moreover, there was no significant difference in tension generated at 50 Hz between old and young animals (p > 0.10 by t test). Increasing age was, however, associated with a decrease in maximal isometric tension expressed in absolute terms (kg/cm2 ) (p < 0.001 by ANOVA) and a reduction in tension at all lengths (p < 0.0001 by ANOVA) (figure 4). The fiber length at which active tension was maximal was unaffected by aging, however (p > 0.1 by t test). Maximal tension at L o was significantly less in muscles from elderly (Le., 18.8 months old) than from young adult (i.e., 4.8 months old) and middle-aged (12.8 months old) animals. However, maximal tension was not significantly different in younger and middle-aged animals (p > 0.25). The twitchltetanus ratio increased significantly with aging (p < 0.001 by ANOVA) and was greater in the older group than in the two younger groups (p < 0.02 by t test for both comparisons). Aging was also associated with a slowing in the twitch tension wave form. Contraction time (CT) and one-half relaxa-
tion time (112 RT) were significantly prolonged (p < 0.0001 by ANOVA for CT and Y2RT) and were greater in the elderly group than in the other two groups (p < 0.02 for both comparisons). There was no difference in CT or Y2RT in diaphragms from the two younger groups of animals, however (p > 0.25 by t test) (table 2). Aging also altered the force-velocity relationship of the diaphragm (figure 5). The velocity of shortening at a given submaximal load (Le., P/P o) decreased with aging (p < 0.0001 by ANOVA for the whole force-velocity curve). Furthermore, the constants a, b, and alP 0 of the Hill equation (table 3) were significantly greater in the older than in the younger animals (p < 0.001 by ANOVA for all comparisons). The maximal velocity of shortening assessed by the slack test (Le., Vos) and by extrapolation of the isotonic force-velocity relationship (Le., Voq) also decreased with aging (p < 0.0001 by ANOVA for both Vos and Voq) (figure 6). Of note, Vos was similar during 50 and 150 Hz stimuli (e.g., 6.25 ± 0.79 SE Lois and 6.44 ± 0.57 SE Lois, respectively; p = 0.69). Maximal velocity of shortening in the entire population of animals correlated weakly with maximal tetanic tension (kg/cm 2 ) at L o (r = 0.32 and 0.41 for Vos
TABLE 2 EFFECT OF AGING ON MAXIMAL TENSION AND THE TWITCH WAVE FORM*
Group Young adult Middle-aged Elderly
Maximal Tension (kg/cm 2 )
L. (mm)
CT (ms)
'hRT (ms)
TwitchiTetanus
2.10 ± 0.10 2.05 ± 0.07 1.70 ± 0.04*
13.8 ± 0.45 14.3 ± 0.31 13.4 ± 0.35
58.60 ± 1.44 59.73 ± 2.13 72.62 ± 1.91 t
54.20 ± 2.23 57.87 ± 2.09 75.54 ± 3.82t
0.49 ± 0.02 0.49 ± 0.01 0.54 ± O.Olt
Definition of abbreviations: L, = optimal length of muscle; CT = contraction time; 'hRT • Values are mean ± SE. t p < 0.02 for comparison with young adult and middle-aged groups.
= one-half
relaxation time.
,
,
0.9 1.1 FIBER LENGTH (L/L ) o
,
1.3
Fig. 4. Length-active tension relationship of the hamster diaphram in young adult (crosses), middle-aged (circles), and elderly (triangles) animals. Symbols represent mean ± SE. Maximal tension at L. is significantly less in muscles from the elderly group than in those from the two younger groups (p < 0.05 for both), but it is not significantly different in animals in the two younger groups (p < 0.25). The fiber length at which active tension is maximum is similar in all three groups (p < 0.1).
and Voq against tetanic tension, respectively; p < 0.05 for both). The Hill coefficient, alP 0, also correlated significantly with maximal tetanic tension (r = 0.49; p < 0.005). Reductions in maximal velocity (Vmax) were also correlated with prolongations in CT and 112 RT (r = - 0.62 and - 0.49 for the relationship of Vos versus CT and Y2RT, respectively, and r = -0.45 and -0.38 for the relationship ofVoq versus CT and Y2RT, respectively; p < 0.01 for all comparisons). Aging also was associated with a decrease in diaphragmatic muscle endurance (p < 0.002 by ANOVA) (figure 7). Tension fell more rapidly in diaphragms from elderly animals than in muscles from animals in the two younger groups, and it was significantly less at the end of the fatigue trial (i.e., at 5 min) in the elderly group when compared with that in the two younger groups (p < 0.01 by t test for both). Discussion
The present study indicates that aging in Golden hamsters is associated with alterations in the contractile properties in the diaphragm. The maximal tensiongenerating ability of the diaphragm is diminished; twitch tension development and relaxation become slower; the forcefrequency relationship is displaced to the left; the force-velocity curve is displaced to the left and assumes a less concave shape; maximal shortening velocity decreases; and the endurance of the di-
1399
AGING AND THE DIAPHRAGM
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Fig. 6. Velocity of shortening at zero load ("max) measured by the slack test method (Vos) (open bar) and by extrapolation of Hill's equation (Voq) (hatched bar) in young, middle-aged, and elderly animals. Symbols represent mean ± SE. "max decreases with aging (p < 0,01 for Vos and p < 0.025 for Voq) and is significantly reduced in the elderly compared with that in the two younger groups (p < 0,01 for both methods).
I
M
AGE
M
FORCE(~P~ Fig. 5. Isotonic force-velocity relationship of the hamster diaphragm in young adult (crosses), middle-aged (circles), and elderly (triangles) animals. Symbols represent mean ± SE. The relationship is less steep (Le., alP. is greater) in the older than in the younger animals. Furthermore, both the extrapolated "max and velocity at submaximalloads are significantly reduced in elder.Iy compared with young adult and middle-aged animals (p < 0,01 for all comparisons).
aphragm in response to subtetanic stimuli is diminished. Interestingly, changes in diaphragm function did not appear to be progressive throughout adulthood. Rather, changes occurred only in the oldest group (i.e., 18.8 months) and were not apparent in 4.9- and 12.8-month-old groups. It is important to point out in this regard that the present study was performed in hamsters over a wide range of the adult life span but before extreme senescence to avoid "survivor" effects caused by attrition in the elderly group. Of note, values for maximal tetanic tension (kg/cm2 ), CT, and YzRT, and extrapolated Vmax obtained in the hamster diaphragm in 4.9- and 12.8-monthold animals resemble those obtained by Metzger and coworkers (11) and Faulkner
Interpretation of Results Displacement of the force-frequency relationship in elderly animals appears to be attributable to prolongation in CT and Yz RT, with greater propensity to develop a fused contraction. Alterations in the force-frequency relationship caused diaphragms from elderly animals to generate a greater percentage of maximal tension at stimulus frequencies of 40 Hz and below, and this is likely to have contributed to the greater rate of fall in diaphragm tension during fatigue trials in these older animals. Isometric tension in response to the 20-Hz stimulus was 89 ± 20/0 SE and 84 ± 2% SEofmaximuminelderly and young animals, respectively (p < 0.05), tending at a given duty cycle (i.e., 0.33) to produce a greater tension-time index during the fatigue trial in muscles from elderly animals (13). Changes in maximal isometric tension and velocity of shortening do not appear to be explained by differences in the
TABLE 3
Correlation Coefficients
Young adult Middle-aged Elderly
0'
o
1
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~~-t-
I
2
I
3
1
4
I
5
TIME (min) Fig. 7. Effect of age on the rate of decline in peak diaphragm tension during repeated contraction in young adult (crosses), middle-aged (circles), and elderly (triangles) animals. Symbols represent mean ± SE. Tension fell more rapidly in diaphragms from elderly animals than in muscles from young adult and middle-aged animals (p < 0.002) and is significantly less at the end of the fatigue trial (Le., 5-min point) in the elderly group than in the two younger groups (p < 0,01 for both).
and colleagues (12) in the rat diaphragm in young adult animals.
EFFECT OF AGING ON THE VELOCITY OF DIAPHRAGM SHORTENING"
Group
"
")~~-a:
o
>
a (g)
b (mm/s)
alP.
6.12 ± 0.56 7.26 ± 0.45 12.09 ± o.oat
6.96 ± o.oa 9.97 ± 0.45 13.11 ± 0.09t
0.16 ± 0.01 0.20 ± 0.02 0.34 ± 0.04t
Definition of abbreviations: Po = maximal isometric tension. " Values are mean ± SE. t p < 0.001 for comparison of all these groups.
force-frequency relationship with aging, however. Tension generated in response to 50 Hz stimuli was 1000/0 of maximum in both young adult and elderly animals. Moreover, maximal velocity of shortening in response to 50 and 150 Hz stimuli was similar, indicating that 50 Hz stimuli achieved full activation during shortening maneuvers in muscles from young and from old animals. In limb muscle, reductions in contractile protein content appear to explain the decrease in maximal tension (14-21). When tension is corrected for alterations in muscle cross-sectional area, however, maximal tension is generally unchanged with aging (14-19), but it may be reduced (20). Although reductions in maximal diaphragmatic tension per unit cross-sectional area in elderly hamsters could represent decreases in the tension-generating ability of the residual myofibrillar material, reductions in maximal tension per unit cross-sectional area of diaphragmatic muscle may simply reflect the presence of noncontractile material that does not contribute to tension development. Detailed examination of muscle structure is needed to test the second possibility. Of note, the shape of the length-tension curve and fiber length at L o were unaffected by aging, which is in agreement with previous results by McCarter and McGee (19) and Brooks and Faulkner (20). The present study also demonstrated that aging is associated with alterations in the force-velocity relationship of the
ZHANG AND KELSEN
1400
diaphragm and the curvature of the forcevelocity relationship, as indicated by the Hill constant, alP o. Reductions in the velocity of diaphragmatic muscle shortening, increases in alP o, and prolongations in CT and Y2RT make the aged diaphragm more closely resemble a slow muscle (22). Changes observed may, therefore, represent alterations in muscle fiber composition in elderly animals. In limb skeletal muscle, aging is associated with a preferential atrophy of fast twitch fibers with the result that slow fibers make up a greater percentage of muscle mass (16,17). There is little information about the effects of aging on fiber composition of the respiratory muscles. We have observed that the cross-sectional area of fast twitch muscle fibers in the intercostal muscles is smaller in 18-month-old hamsters, and a greater percentage of the muscle mass is composed of slow twitch muscle fibers compared with the muscle mass in 12month-old animals (23). The changes in fast twitch fiber size in the intercostal muscles, therefore, appear to be similar to what has been observed in limb muscle. Changes in fiber composition, however, do not appear to be the sole explanation for the effects of aging on diaphragmatic muscle function for several reasons. First, the maximal velocity of unloaded shortening assessed by the slack test is decreased. Values obtained in the slack test appear to be determined by the rate of shortening of the most rapidly shortening fibers, Le., fast twitch fibers (24), suggesting that the velocity of shortening of the fast fiber population is diminished. Second, recent studies indicate that the contraction and relaxation time of individual fast twitch and slow twitch motor units in the soleus and extensor digitorum longus muscles increase with aging (25). Reductions in the speed of shortening and tension kinetics of diaphragm muscle bundles, therefore, may be a reflection of changes in the properties of individual fast and slow motor units. Third, the reduction in the endurance of the diaphragm in elderly animals is not explainable by preferential fast fiber atrophy since such a change in fiber composition should have increased endurance.
The Effect of Aging on Limb Skeletal Muscle A large body of literature obtained from studies on limb skeletal muscles in a variety of species indicates that aging is associated with reductions in muscle
weight, fiber diameter and fiber number, myofibrillar protein content, and the ability to synthesize high-energy phosphates (1,2,3, 16, 19,20,25). Aging also appears to be associated with subtle changes in the structure and function of the motor unit and myoneural junction (3,4). Although it is quite clear that aging per se influences the structure and biochemical properties of limb skeletal muscle, recent studies indicate that there are surprisingly few changes in maximal tension, shortening velocity, and endurance of limb-skeletal muscles excited electrically in vitro (14, 15, 19). For example, maximal active tension (Le., kg/cm 2 ) of the soleus, a slow muscle (14, 15, 19), the extensor digitorum longus (EDL), a fast muscle (14, 15), and the omohyoideus, a mixed muscle (19), is similar in young adult (Le., 9 months old) and elderly (Le., 28 to 30 months old) rats. In fact, Eddinger and coworkers (14) found that maximal tension per unit of cross-sectional area in single fibers of the soleus was greater in 30-month-old than in 9-month-old rats. On the other hand, Brooks and Faulkner (20) noted a 22010 reduction in maximal tension in whole muscle bundles of the EDL in elderly mice. (Of note, maximal soleus tension was unaffected by aging [20].) Similarly, there appears to be no reduction in Vmax determined either by the slack test (14) or by extrapolation of the isotonic force-velocity relationship (14, 15, 20) in the soleus or the EDL (20). In fact, Eddinger and coworkers (14) observed an increase in Vmax determined by the slack test in soleus muscle bundles and skinned single fibers. On the other hand, extrapolated Vmax and isotonic velocity of shortening at submaximal loads (Le., PI Po 0.1 to 0.9) of the EDL was reduced in 30-month-old compared with 9-month-old animals (14). Finally, several studies have observed no effect of aging on the endurance of the soleus (14, 19), EDL (14), or omohyoideus (19) muscles during electrically stimulated contractions in vitro. The above results on the effects of aging on maximal tension, shortening velocity, and endurance in limb skeletal muscles are in contrast to the results we obtained in the diaphragm where large reductions in performance were observed. Respiratory skeletal muscle, like skeletal muscle in other parts of the body, is highly plastic and alters its structure, Qiochemical properties, and contractile function in response to alterations in applied load, precontraction length, and
pattern of activity (6, 27). Aging is associated with changes in the mechanics of breathing, lung gas exchange, and chemosensitivity to hypoxia and hypercapnia, which may alter the load and level of activity of the diaphragm (28-30). Moreover, the activity level of older animals may be less than that of younger animals. Reductions in the level of ventilation in older compared with younger animals (as a result of reductions in physical activity or reductions in respiratory motor activity at a given level of physical activity) could have contributed to the changes in muscle function observed. However, we know of no data in which respiratory motor activity has been compared in young and old animals. Regardless of mechanism(s), the results of the present study are likely to apply to humans. For example, decreases in maximal diaphragmatic tension with aging in the hamster appear to have a similar time course and magnitude to reductions in inspiratory muscle strength assessed from maximal static inspiratory pressure in normal human subjects (1). Maximal static inspiratory pressure in elderly (Le., 80-yr-old) male and female subjects is approximately 30% lower than in young (i.e., 20- to 30-year-old) male and female subjects and does not appear to decrease significantly with aging until after approximately 50 to 60 yr of age (reference 1 and figures 2 and 3). References 1. Black LF, Hyatt RE. Maximal respiratory pressures: normal values and relationship to age and sex. Am Rev Respir Dis 1968; 99:696-702. 2. Larsson L, arimby 0, Karlsson J. Muscle strength and speed of movement in relation to age and muscle morphology. J Appl Physiol 1979; 451-6. 3. Brown WE A method for estimating the number of motor units in thenar muscles and the changes in motor unit count with aging. J Neurol Neurosurg Psychiatry 1972; 35:845-52. 4. Banker BQ, Kelly SS, Robbins, N. Neuromuscular transmission and correlative morphology in young and old mice. J Physiol (Lond) 1983; 339: 355-75. 5. Chesky JA. Comparative aspects of declining muscle function. Aging 1978; 6:179-200. 6. Supinski as, Kelsen so. Effect of elastaseinduced emphysema on the force generating ability of diaphragm. J Clin Invest 1982; 70:978-88. 7. Kelsen SO, Ference M, Kapoor S. Effects of prolonged undernutrition on structure and function of the diaphragm. J Appl Physiol 1985; 58:1354-9. 8. Orsini MW. Inbred and genetically defined strains of laboratory animals. Part 2. Hamster, guinea pig, rabbit and chicken. In: Altman PL, Katz DD, eds. Biological handbooks III. Federation of American Societies for Experimental Biology, 1979; 436-7. 9. Hill AV. The heat of shortening and dynamic
AGING AND THE DIAPHRAGM
constants of muscle. Proc R Soc London [Bioi] 1938; 126:136-95. 10. Edman KAP. The velocity of unloaded shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibers. J Physiol (Lond) 1979; 291:143-59. 11. Metzger JM, Schmidt KB, Fitts RH. Histochemical and physiological characteristics of the rat diaphragm. J Appl Physiol 1985; 58:1085-91. 12. Faulkner JA, Claflin DR, McCullyKK, Jones AD. Contractile properties of bundles of fiber segments from skeletal muscles. Am J Physiol 1982; 243:C66-73. 13. Kelsen SG, Nochomovitz ML. Fatigue of the mammalian diaphragm in vitro. J Appl Physiol 1982; 53:440-7. 14. Eddinger n, Cassens RG, Moss RL. Mechanical and histochemical characterization of skeletal muscles from senescent rats. Am J Physiol 1986; 251:C421-30. 15. Fitts RH, Troup JP, Witzmann FA, Holloszy JO. The effect of aging and exercise on skeletal muscle function. Mech Ageing Dev 1984; 27:161-72. 16. Gutmann E, Hanzlikova V, Vyskocil F. Age changes in cross striated muscle of the rat. J Phys-
1401 iol (Lond) 1971; 219:331-43. 17. Gutmann E, Syrovy I. Contraction properties and myosin ATPase activity of fast and slow senile muscles of the rat. Gerontology 1974; 20:239-44. 18. Larsson 1. Aging in mammalian skeletal muscle. In: Mortimer JA, Pirozzolo FJ, Maletta GJ, eds. The aging motor system. New York: Praeger, 1982; 61-97. 19. McCarter R, McGee MRM. Influence of nutrition and aging on the composition and function of rat skeletal muscle. J Gerontol1987; 42:432-41. 20. Brooks SV, Faulkner JA. Contractile properties of skeletal muscles from young, adult and aged mice. J Physiol (Lond) 1988; 404:71-82. 21. Daw CK, Starnes JW, White TP. Muscle atrophy and hypoplasia with aging: impact of training and food restriction. J Appl Physiol 1988; 64: 2428-32. 22. Ranatunga KW. The force-velocity relation of rat fast- and slow-twitch muscles examined at different temperatures. J Physiol (Lond) 1984; 351:517-29. 23. Bao S, Thomas AJ, Kelsen SG. Structure of intercostal muscle: topographic and age-dependent effects (abstract). Fed Proc 1987; 43:818. 24. Dennis RC, Faulkner JA. Shortening veloci-
ty extrapolated to zero load and unloaded shortening velocity of whole rat skeletal muscle. J Physiol (Lond) 1985; 359:357-63. 25. Edstrom L, Larson 1. Effects of age on contractile and enzyme-histochemical properties of fastand slow-twitch single motor units in the rat. J Physiol (Lond) 1987; 392:129-45. 26. Moller P, Bergstron J, Furst P, Hellstrom K. Effect of aging on energy-rich phosphagen in human skeletal muscles. Clin Sci 1980; 58:553-5. 27. Jolesz F, Sreter FA. Development, innervation and activity-pattern induced changes in skeletal muscle. Annu Rev Physiol 1981; 43:531-52. 28. Kronenberg RS, Drage CWo Attenuation of the ventilatory and heart rate responses to hypoxia and hypercapnia with aging in normal men. J Clin Invest 1973; 52:1812-9. 29. Holland J, Milic-Emili J, Macklem PT, Bates DV. Regional distribution of pulmonary ventilation and perfusion in elderly subjects. J Clin Invest 1968; 47:81-92. 30. Turner JM, Mead J, Wohl ME. Elasticity of human lungs in relation to age. J Appl Physiol1968; 25:664-71.
