We examined single muscle fiber contractile function of the oldest-old (3F/2M, 89 ± 1 yr old) enrolled in The Health, Aging, and Body Composition Study (The Health ABC Study). Vastus lateralis muscle biopsies were obtained and single muscle fiber function was determined (n = 105) prior to myosin heavy chain (MHC) isoform identification with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cross-sectional area of MHC I muscle fibers (5,576 ± 333 μm2; n = 58) was 21% larger (P < 0.05) than MHC IIa fibers (4,518 ± 386 μm2; n = 47). Normalized power (an indicator of muscle fiber quality incorporating size, strength, and speed) of MHC I and IIa muscle fibers was 2.3 ± 0.1 and 17.4 ± 0.8 W/l, respectively. Compared with previous research from our lab using identical procedures, MHC I normalized power was 28% higher than healthy 20 yr olds and similar to younger octogenarians (∼80 yr old). Normalized power of MHC IIa fibers was 63% greater than 20 yr olds and 39% greater than younger octogenarians. These comparative data suggest that power output per unit size (i.e., muscle quality) of remaining muscle fibers improves with age, a phenomenon more pronounced in MHC IIa fibers. Age-related single muscle fiber quality improvements may be a compensatory mechanism to help offset decrements in whole muscle function.
- single fiber
- muscle quality
- contractile function
NEW & NOTEWORTHY
This is the first study to report single muscle fiber strength, speed, and power in the oldest-old. These data suggest an improvement in myocellular quality with age that is more pronounced in the fast-twitch muscle fibers. The improved quality of these remaining muscle fibers suggests a “survival of the fittest” phenomenon that is in contrast to the classic aging skeletal muscle dogma.
the 2010 U.S. census projects the number of people in the oldest-old age group (those age 85 yr old and older) will triple from 5.8 to 19 million by 2050 (71). A hallmark characteristic of advancing age is a decline in the number (41) and size (67) of muscle fibers contributing to a loss of muscle mass and strength in older adults (25). Impaired muscle function is related to mobility impairment (15), disability (29), and loss of independence (52). A more definitive understanding of the physiological mechanisms underlying age-related skeletal muscle decline may help guide therapeutic interventions to ameliorate mobility limitations, subsequently enhancing quality of life in the expanding demographic of the oldest-old.
Age-associated muscle mass declines (3-8% per decade) begin during the fourth decade of life (14, 42, 60) and accelerate after the age of 75 yr (72). Decreases in muscle mass with age are suggested to contribute to a loss of muscle strength and, perhaps more importantly, muscle power (17, 28, 54). However, muscle atrophy has been proposed to account for only half of the lower peak muscle power observed in elderly individuals (63). This suggests a loss in whole muscle quality (power output per muscle cross-sectional area) during the aging process. Muscle power is shown to decline earlier (beginning as early as 20 yr of age) and 10% more per decade than muscle strength with aging (46). Furthermore, low muscle power is associated with a 2- to 3-fold greater risk for mobility limitation compared with low muscle strength (4). Whole muscle quality reductions in older individuals could be due to a number of factors such as fiber type transition (39, 42), alterations in neuromuscular function (11, 66), excitation-contraction uncoupling (51), fat infiltration (24), or alterations to the intrinsic properties of the individual muscle fibers (40).
Investigations of the cellular response to the aging process provide unique insights into age-related muscle dysfunction. Preliminary studies inspecting single muscle fiber quality in aged rodents reported 20–25% lower single fiber specific force (force adjusted for size) compared with young animals (6, 43, 64). These findings were corroborated by early aging human single fiber studies (19, 40, 50), suggesting that age-related alterations at the cross-bridge level contribute to whole muscle quality decline. However, recent human investigations contrast these early reports showing a preservation of single fiber specific force with age (18, 36, 53, 61). While the reasons for the differences among the early and more recent studies are not clear, we suspect it may be related to overall fiber yield (i.e., the number of fibers studied per individual) in combination with different physical activity levels between older and younger participants studied (9, 67).
The purpose of this investigation was to evaluate single muscle fiber quality in slow- and fast-twitch muscle fibers of a unique ageing cohort (>85 yr of age). Our primary interest was normalized power (a measure accounting for size, strength, and speed) since this provides an integrated performance index incorporating quantitative and qualitative aspects of contractile function. We also assessed specific force to complement normalized power as an additional measure of muscle quality and to compare our findings to previous literature.
Five healthy independently living older adults, 3 females and 2 males (Table 1), were recruited as part of The Health, Aging, and Body Composition Study (Health ABC Study) and included in the analysis. All subjects were over 85 yr of age (87–90 yr), nonexercising, nonobese, and otherwise healthy. Before participation in the study, subjects were informed of all risks and procedures, and written informed consent was obtained. Institutional Review Boards approved all procedures at the participating institutions in a manner consistent with the Declaration of Helsinki.
Resting muscle biopsies (5) were obtained from the vastus lateralis (VL) of each subject. Muscle samples were sectioned longitudinally into several pieces and placed in cold skinning solution prior to being stored at −20°C for later analysis of single muscle fiber physiology. Following a single muscle fiber experiment, each fiber was analyzed for MHC composition, as described in Fiber type analysis.
Skinning, Relaxing, and Activating Solutions
The skinning solution contained (in mM) 125.0 K propionate, 2.0 EGTA, 4.0 ATP, 1.0 MgCl2, 20.0 imidazole (pH 7.0), and 50% (vol/vol) glycerol. The compositions of the relaxing and activating solutions were calculated using an interactive computer program described by Fabiato and Fabiato (16). These solutions were adjusted for temperature, pH, and ionic strength using stability constants in the calculations (22). Each solution contained (in mM) 7.0 EGTA, 20.0 imidazole, 14.5 creatine phosphate, 1.0 free Mg2+, 4.0 free MgATP, KCl, and KOH to produce an ionic strength of 180 mM and a pH of 7.0. The relaxing and activating solution had a free [Ca2+] of pCa 9.0 and pCa 4.5, respectively (where pCa = −log [Ca2+]).
Single Muscle Fiber Experimental Set-Up
On the day of an experiment, a 2.5- to 3.0-mm muscle fiber segment was randomly isolated from a muscle bundle and transferred to an experimental chamber filled with relaxing solution where the ends were securely fastened between a force transducer (model 400A, Cambridge Technology, Lexington, MA) and a direct-current torque motor (model 308B, Cambridge Technology) as described by Moss (48). The force transducer and torque motor were calibrated before each experiment. Instrumentation was arranged so a muscle fiber could be rapidly transferred back and forth between experimental chambers filled with relaxing (pCa 9.0) or activating (pCa 4.5) solutions. The apparatus was mounted on a microscope (Olympus BH-2, Japan) to view the fiber (×800) during an experiment. Using an eyepiece micrometer, sarcomere length along the isolated muscle was adjusted to 2.5 μm, and the fiber length (FL) was measured (69). All single fiber experiments were performed at 15°C.
Unamplified force and length signals were sent to a digital oscilloscope (Nicolet 310, Madison, WI), enabling monitoring of muscle fiber performance throughout data collection. Analog force and position signals were amplified (Positron Development, Dual Differential Amplifier, 300-DIF2, Inglewood, CA), converted to digital signals (National Instruments, Austin, TX), and transferred to a computer (Micron Electronics, Nampa, ID) for analysis using customized software. Servomotor arm and isotonic force clamps were controlled using a computer-interfaced force-position controller (Positron Development, Force Controller, 300-FC1, Inglewood, CA).
For each single muscle fiber experiment, a fiber with a compliance (calculated as FL divided by y-intercept) >10% and/or a decrease in peak force (Po) of >10% was discarded and not used for analysis. The within-fiber test/retest of a single muscle fiber in our lab for the measurements of size, force-power relationships, Po, and contractile velocity was <1%. The coefficients of variation for the force transducer and servomechanical lever mechanism during the timeframe of this investigation was <1%. Following completion of single muscle fiber physiology experiments, each fiber was solubilized in 80 μl of 1% SDS sample buffer and stored at −20°C until assayed for MHC fiber type.
Single Muscle Fiber Analysis
Individual muscle fibers were analyzed for force-velocity relationships and power, peak force (Po), maximal unloaded shortening velocity (Vo), diameter, and fiber type. Experimental procedures were identical to those previously used in our human studies (67, 69).
Single muscle fiber power.
Submaximal isotonic load clamps were performed on each fiber for determination of force-velocity parameters and power. Each fiber segment was fully activated in pCa 4.5 solution and subjected to a series of three isotonic load steps. This procedure was performed at various loads so that each fiber underwent a total of 15–18 isotonic contractions.
For the resultant force-velocity relationships, load was expressed as P/Po (P = force during load clamping, Po = peak isometric force developed before submaximal load clamps). Force and shortening velocity data points were derived from the isotonic contractions and fit by the hyperbolic Hill equation (32). Only individual experiments in which R2 was ≥0.98 were included for analysis.
Fiber peak power was calculated from the fitted force-velocity parameters (Po, Vmax, and a/Po, where a is a force constant and Vmax is the y-intercept). Absolute power (μN·FL/s) was defined as the product of force (μN) and shortening velocity (FL/s). Normalized power (W/l) was defined as the product of normalized force and shortening velocity.
Single muscle fiber Po.
Force and position transducer outputs were amplified and sent to a microcomputer via a Lab-PC+ 16-bit data acquisition board (National Instruments, Austin, TX). Resting force was monitored and then the fiber was maximally activated in pCa 4.5 solution. Peak active force (Po) was determined in each fiber by computer subtraction of the baseline force from the peak force in the pCa 4.5 solution.
Single muscle fiber Vo.
Fiber unloaded shortening velocity (Vo) was measured by the slack-test technique as described by Edman (13). The fiber was fully activated in pCa 4.5 solution and rapidly released to a shorter length, such that force fell to baseline. The fiber shortened, taking up slack, after which force began to redevelop. Then the fiber was placed in pCa 9.0 solution and returned to original length. Computer analysis determined the duration of unloaded shortening, or time between onset of slack and redevelopment of force. Four different activation and length steps (150, 200, 250, and 300 μm; ≤15% of FL) were used for each fiber, with the slack distance plotted as a function of the duration of unloaded shortening. Fiber Vo (FL/s) was calculated by dividing the slope of the fitted-line by the fiber segment length (data were normalized to a sarcomere length of 2.5 μm).
Single muscle fiber diameter.
A video camera (CCD-IRIS, DXC-107A; Sony, Japan) connected to the microscope and computer interface allowed for viewing and storage of single muscle fiber digital images. Fiber diameter was determined from an image taken with the fiber briefly suspended in air (<5 s). Fiber width (diameter) was determined at three points along the segment length of the captured image using NIH public domain software (Scion Image, release Beta 4.0.2, for Windows). For the fiber size-dependent variables (i.e., Po/CSA and normalized power), CSA was determined with the assumption that the fiber forms a cylindrical shape while suspended in air.
Fiber type analysis.
Following the single fiber contractile measurements, the MHC isoform profile was analyzed for each fiber segment using SDS-PAGE. Briefly, samples were run overnight at 4°C on a Hoefer SE 600 gel electrophoresis unit (San Francisco, CA) utilizing a 3.5% (wt/vol) acrylamide stacking gel with a 5% separating gel (74). After electrophoresis, gels were silver stained as described by Giulian et al. (21). MHC isoforms (I, I/IIa, IIa, IIa/IIx, IIx, I/IIa/IIx) of each single muscle fiber were identified according to migration rate as we have previously described (74). A MHC gel image from a Health ABC Study participant is shown in Fig. 1.
Single muscle fiber size and performance characteristics from the Health ABC Study participants are presented as means ± SE. Potential differences between size and contractile characteristics of MHC I and IIa fibers were analyzed using dependent two-tailed Student's t-tests with a Bonferroni adjustment for contractile measures. As a result of the descriptive nature of the study, exploratory data analysis (70) was used to examine and compare these data to previous investigations of single muscle fiber function and size.
Single Muscle Fiber Power
The normalized power values of individual MHC I and IIa muscle fibers from The Health ABC Study participants are shown in Fig. 2 alongside individual muscle fibers from two younger cohorts. Additional normalized power comparisons among various healthy, athletic, and unique (spinal cord injured and champion sprint runner) cohorts are shown in Fig. 3. MHC IIa single muscle fibers were 5× more powerful (P < 0.01) than MHC I fibers (Table 2). When power was normalized to cell size, the MHC IIa fibers (17.4 ± 0.8 W/l) were 7.5× more powerful (P < 0.01) than MHC I fibers (2.3 ± 0.1 W/l).
Single Muscle Fiber Force and Velocity
There was no difference in peak force (Po) between MHC I and IIa muscle fibers (Table 2). When peak force was adjusted to fiber cross-sectional area (Po/CSA: specific force), specific force of MHC IIa fibers was 41% greater (P < 0.01) than MHC I fibers. Contractile speed (Vo and Vmax) of the MHC IIa muscle fibers was ∼4-fold faster (P < 0.01) than MHC I fibers.
Single Muscle Fiber CSA
Single muscle fiber size data are summarized in Table 2. Cross-sectional area (CSA) of MHC I muscle fibers was 21% larger (P < 0.05) than MHC IIa fibers.
Single Muscle Fiber Analysis
The data presented from Health ABC Study participants were obtained from a total of 123 single muscle fibers. Of these, 105 single fibers were included in the analysis (58 MHC I and 47 MHC IIa). No fibers containing the MHC IIx isoform were found (MHC IIx, IIa/x, or I/IIa/IIx), and only 6 hybrid MHC I/IIa fibers were identified and were thus not included in the analysis. There were 12 additional fibers not included in the analysis due to failed single fiber experiments (n = 11) or inability to be typed with SDS-PAGE (n = 1), a result of low or missing protein in the SDS buffer tube. The low failure rate (<10%) of the single muscle fiber physiology experiments is an indication of the structural integrity of the muscle fibers studied from this cohort and thus reflective of the myocellular characteristics of these individuals.
Our comparative results suggest an improvement in single muscle fiber quality with advancing age, especially in MHC IIa fibers. It appears that myocellular and whole muscle quality in humans exhibit disparate responses to the aging process (55). Single muscle fiber quality improvements may serve as a compensatory mechanism to help attenuate deficits in whole muscle size and performance seen in aging individuals (23, 25).
Our laboratory previously reported comparable MHC IIa quality in a cohort of octogenarian and younger sedentary women (53). Comparison of MHC IIa normalized power data from the oldest-old with fibers from healthy 20 and 80 yr olds suggests that single fiber quality is not only preserved but appears to increase with advancing age (Fig. 2B). A typical normalized power output of a MHC IIa muscle fiber is ∼5–12 W/l across various age groups from our laboratory (68) and others (45, 55, 73). Previously, we indicated that 10 W/l represents an average value for MHC IIa normalized power in a large sample of older men and women (70–82 yr olds) (61). This value is greater than what is seen (Fig. 3B) in MHC IIa fibers from young (∼20 yr old) and middle age (∼40 yr old) individuals (6–8 W/l) engaged in vigorous aerobic exercise training (31, 73). Therefore, we interpret a MHC IIa muscle fiber producing >10 W/l to have above-average fiber quality. Ninety-eight percent of the MHC IIa single muscle fibers from the oldest-old had normalized power values above this threshold (Fig. 2B). When extended to a normalized power value > 20 W/l, more than 25% of the MHC IIa fibers from the oldest-old were above this value compared with zero from the young cohort and 5% from younger octogenarians (61).
It has been suggested that the greater single muscle fiber quality observed in elderly individuals is a product of a “survival of the fittest” phenomenon, in which only the best fibers are preserved as fiber number declines (18, 45, 55, 56). Survival of the highest quality muscle fibers is similarly proposed to occur in spinal cord injured (SCI) patients, who undergo significant muscle remodeling due to an absence of neuromuscular recruitment (45) (Fig. 3). However, a number of MHC IIa fibers from the oldest-old produced normalized power values far greater than were observed in any younger individuals (67) or even a champion sprint runner (17.1 ± 0.5 W/l) (68). Thus it seems some adaptive mechanism exists to complement the survival of the fittest and further enhance muscle fiber quality in these elderly individuals.
The superior normalized power of MHC IIa fibers in the oldest-old is in close agreement with the greater specific force-producing capacity of these fibers [42% greater than 20 yr olds and 19% greater than younger octogenarians (61, 67)]. Enhanced muscle quality in MHC IIa fibers of elderly individuals may be related to fiber atrophy, which is characteristic of aging muscle. Greater specific force production in smaller MHC IIa fibers of elderly individuals (∼70 yr olds) was recently observed by Miller et al. (47), who attributed this difference to an age-related slowing of cross-bridge kinetics leading to greater myofilament stiffness and enhanced force transmission. Sluggish cross-bridge kinetics in atrophying MHC IIa fibers may be explained by increased packing density of contractile proteins which is postulated to prolong cross-bridge attachment and increase internal drag (57). Inconsistent with this proposition, MHC IIa contractile velocity of the oldest-old was no slower than that of healthy 20 yr olds (67). This suggests that the MHC IIa fibers in this cohort have developed unique architectural arrangements to overcome the greater internal drag suspected to occur with atrophy (8). Molecular alterations to regulate myosin light chain composition and/or phosphorylation status may also help to attenuate the slower shortening velocity observed in previous aging single fiber investigations (37, 40). Collectively, these aging single muscle fiber comparisons suggest that atrophy with minimal changes in absolute force and shortening velocity contributes to improvements in MHC IIa single fiber quality of the oldest-old.
Equivalent MHC I fiber quality has been demonstrated by previous research from our lab in younger (∼20 yr old) and older (∼80 yr old) men and women (67). Normalized power of MHC I fibers from the oldest-old, a cohort 10 years older than the aforementioned older cohort, was 28% greater than healthy 20 yr olds (67) and similar to younger octogenarians (61) (Fig. 2A). Similar to what was seen in MHC IIa fibers, greater MHC I normalized power is in agreement with improvements in specific force (32% greater in the oldest-old than healthy 20 yr olds) (67). The comparatively modest difference in MHC I quality between younger and older individuals has been shown previously (47), and may be due to the preservation or compensatory increase in MHC I fiber size observed with aging (3, 56). Dissimilar to what was seen in MHC IIa fibers, unloaded shortening velocity of the slow (MHC I) fibers was lower in elderly individuals (34% less than healthy 20 yr olds) (67). Depressed shortening velocity of MHC I fibers is generally a characteristic of habitual use (such as distance running) and has been observed by previous aging single fiber investigations (18, 31, 47). Taken together, these cross-sectional findings suggest that MHC I quality improvements are less than that of MHC IIa muscle fibers, likely due to maintenance of MHC I fiber size and a reduction in shortening velocity.
As a complement to the greater single muscle fiber quality in the oldest-old, very few (<5%) MHC-coexpressing muscle fibers were identified. This is in contrast to previous aging literature (35, 74) demonstrating a far greater (∼30%) occurrence of hybrid fibers in aging muscle. Considerable proportions (∼50%) of MHC-coexpressing muscle fibers have also been observed with extreme disuse such as paralysis (45) and following 90 days of bed rest (20). The small hybrid fiber population observed in aging athletes (1, 34) in conjunction with the substantial reduction of MHC coexpression demonstrated in older adults after exercise training (74) and spinal cord injured patients with functional electrical stimulation (2) suggests that frequent use curtails hybrid fiber expression. Although the number of fibers sampled was relatively small, perhaps the paucity of MHC-coexpressing muscle fibers observed in the oldest-old is a result of apoptotic eradication of hybrid fibers (58). Frequent recruitment of remaining fibers could then explain the lack of MHC coexpression observed in the oldest-old. Thus the substantial fiber loss combined with the habitual physical activity status associated with independently performing tasks of daily living in these individuals may serve as a stimulus to maintain and/or improve the contractile function as well as the phenotype of surviving fibers.
Interpretation of the present findings in light of previously published literature highlights that, while aging appears to have a positive impact on single muscle fiber quality in independent free-living humans, aging rodents demonstrate an inability to preserve contractile protein content (65) and function (43), resulting in lower single fiber quality (33, 44). At the whole muscle level, however, aging is shown to result in a progressive deterioration of both muscle mass and function in humans and animals (49). In aging humans, whole muscle atrophy is mediated by a reduction in fiber number (∼50% from 20 to 80 yr of age) and fast-fiber specific atrophy with a gradual compensatory improvement in quality of remaining fibers (18, 27, 42). In contrast, in the oldest-old rats (30 mo) (59), whole muscle atrophy occurs without a loss of muscle fibers (7, 12), and a decrease in single fiber size and quality is observed (33, 64). Collectively, these data suggest that although findings at the whole muscle level are similar between aging humans and rodents, it appears the underlying muscle biology regulating fiber number and quality between species is perhaps different. This hypothesis is not unprecedented, as additional evidence for interspecies differences in muscle regulation can be found in the absence of the MHC IIb isoform in humans (62). Differential regulation of myocellular size and quality in humans and rodents may be related to the relatively longer duration of the aging process between species (60 yr vs. 18 mo, respectively) but currently remains unresolved and warrants further discussion and research.
We had a unique opportunity within the Health ABC project to examine muscle function at the cell level in the oldest-old. Obvious limitations to our data set involve the modest sample size for these types of measurements and cross-sectional comparisons. Gaining access to independent living healthy volunteers in this age range is challenging. The homogeneity of this new data set provides merit for our interpretation and guidance for future investigations. We were also limited in the amount of muscle tissue allocated for these single muscle fiber contractile function measurements. Based upon these findings, additional research on the proteins involved in the contractile process with aging is warranted. Indeed, a new paradigm is beginning to emerge that suggests multiple components of skeletal muscle remain intact (26, 53, 67) or are improved with aging beyond the 8th decade of life (47, 56). Very recent evidence suggests calcium kinetics (38) and mitochondrial function (10) may also track a similar pattern to the contractile function measurements presented here.
To our knowledge, this study was the first to examine single muscle fiber strength, speed, and power in the oldest-old (mean age > 85 yr old). Comparison of single muscle fiber data from this unique survival subset of individuals to various younger populations builds upon previous aging investigations from our lab (53, 61, 67) and others (18, 47, 55) corroborating a continued improvement in muscle fiber quality with advancing age, particularly in MHC IIa fibers. Myocellular quality improvements in surviving MHC IIa fibers appear to be largely a product of preserved contractile function in the presence of substantial fiber atrophy. Additional examinations of the myocellular proteome as well as other aspects of single fiber integrity (mitochondrial capacity and/or fiber architecture) may provide further insight into the cellular adaptations of skeletal muscle in response to the aging process.
This research was supported by National Institute on Aging (NIA) contracts N01-AG-6-2101, N01-AG-6-2103, N01-AG-6-2106; NIA Grants R01-AG-028050 and R01-AG038576; and NINR Grant R01-NR012459. This study was supported in part by the Intramural Research Program of the NIA.
No conflicts of interest, financial or otherwise, are declared by the author(s).
G.J.G., R.S., K.A.M., U.R., K.M., and B.H.G. performed experiments; G.J.G., K.A.M., and S.W.T. analyzed data; G.J.G., K.A.M., U.R., and S.W.T. interpreted results of experiments; G.J.G., K.A.M., and S.W.T. prepared figures; G.J.G. and S.W.T. drafted manuscript; G.J.G., R.S., K.A.M., U.R., K.M., P.C., A.B.N., S.C., T.H., S.K., B.H.G., and S.W.T. approved final version of manuscript; R.S., K.A.M., U.R., K.M., P.C., A.B.N., S.C., T.H., S.K., B.H.G., and S.W.T. edited and revised manuscript; B.H.G. and S.W.T. conception and design of research.
We thank Dr. Gwenaelle Begue for technical assistance with single muscle fiber classification.