Force-Generation Capacity of Single Vastus Lateralis Muscle Fibers and Physical Function Decline With Age in African Green Vervet Monkeys

Abstract

Previous studies on the contractile properties of human myofibrils reported increase, decrease, or no change with aging, perhaps due to the differences in physical activity, diet, and other factors. This study examined physical performance and contractile characteristics of myofibrils of vastus lateralis (VL) muscle in young adult and old African green vervet monkeys. Animals were offered the same diet and lived in the same enclosures during development, so we were able to examine skeletal muscle function in vivo and in vitro with fewer potential confounding factors than are typical in human research studies. Fiber atrophy alone did not account for the age-related differences in specific force and maximal power output. Regression modeling used to identify factors contributing to lower fiber force revealed that age is the strongest predictor. Our results support a detrimental effect of aging on the intrinsic force and power generation of myofilament lattice and physical performance in vervet monkeys.

Loss of mass and force and slower shortening velocity are hallmarks of aging skeletal muscle (1–4). Loss of fibers and selective fast-fiber atrophy (5–7), excitation–contraction uncoupling (8,9), and perturbations to cross-bridge function (10–12) seem to account for the reduced ability of whole muscle to produce force under isometric or shortening conditions in old age.

Even though this loss in muscular function with aging is widely accepted, the intrinsic capacity of single muscle fiber to sustain force throughout the lifetime of higher mammals, including humans, is still uncertain. Several studies have reported a significant reduction in the contractile properties of skinned single muscle fiber with aging, including reduced maximal isometric force (P _o), normalized force to fiber cross-sectional area (CSA; P _o/CSA), and unloaded shortening velocity (V _o; 11,13–16). These reports indicate that both the size and quality of individual fibers decrease with age. The proposed mechanisms are either fewer strongly bound cross-bridges during maximal activation or the force-generating ability of each cross-bridge is reduced (11,13).

In contrast, a number of aging studies have reported preserved single muscle fiber contractile properties (17–20) or even a slight increase in type I and IIa fibers’ maximal force (21,22) and conclude that the intrinsic properties of cross-bridge mechanics are preserved with aging. These contrasting results could derive from diverse factors influencing muscle fiber structure and/or function, such as variation in the subject nutrition, genetics, and history of physical activity (23,24). Furthermore, the participants’ diverse education, occupational history, and socioeconomic status complicate interpretation of various outcomes (25).

Recently, we developed and applied a battery of physical performance tests modeled after human interventional studies to various monkey species (26). Identifying relationships between physical function and the functional and biomolecular characteristics of single muscle fibers could provide valuable insights into the mechanistic bases of functional decline with aging.

The goal of this work was to study age-dependent changes in the intrinsic contractile properties of myofilaments and physical performance in African green monkeys subjected to the same diet and living in the same indoor and outdoor enclosures. We hypothesized that diminished skeletal muscle function in vitro is associated with the impaired physical performance that often accompanies aging and that measuring it in the vastus lateralis (VL) from monkeys could reduce the number of potential confounding factors that typically arise in human research studies.

Methods

Participants

The study included eight adult female African green vervet (Chlorocebus aethiops sabaeus) monkeys: four young adults (11±1 year), corresponding roughly to human beings in their thirties, and four old adults (23±1 year), corresponding roughly to human beings in their seventies. All participants were born at the original Vervet Research Colony at University of California, Los Angeles, and raised in social groups managed to reflect the natural social composition of vervet groups in the wild. Animals were descendants of 57 wild founders (29 females and 28 males) captured in St. Kitts and Nevis, West Indies, and introduced into the colony between 1975 and 1983. Animals were transferred in 2007 to the Primate Center at Wake Forest University, where the current studies were performed. All had free access to standard commercial monkey chow (Purina LabDiet) and water. To be included in the physical function studies, animals had to be in good health, with normal posture and locomotion patterns, and not known to be pregnant or to have offspring younger than 9 months of age. All of the monkeys lived in stable social groups of 11–49 in housing units with approximately 28 m² indoors and 111 m² outdoors. They were observed in the outdoor section, which was furnished with perching and climbing structures that enabled all the locomotor behavior reported here. After behavioral observations were complete, the monkeys were moved to pair caging for 1 year prior to tissue collection. All procedures involving monkeys were conducted in accordance with state and federal laws, standards of the Department of Health and Human Services, and guidelines established by the institutional Animal Care and Use Committee.

Body Weight, Body Mass Index, and Body Composition

Body weight and body mass index were measured once during the time period of the behavioral performance observations. Body mass index was calculated as body mass (kg)/trunk length (m), where trunk length extends from the crown to pubic symphysis. Body composition was determined from whole scans of animals using a Hologic Discovery A dual energy X-ray densitometer just prior to necropsy.

Physical Performance Observation

All physical performance was recorded by observers experienced in recording nonhuman primate behavior. Interobserver reliability was r ≥ .92. Methods used to assess physical performance have been described in detail (26) and are described briefly later. Physical performance tests do not necessarily correspond to maximal performance recorded in humans.

Usual Walking Speed

All monkeys were observed ad libitum for documentation of their usual walking speed following standardized criteria (27). Visible points on various structures throughout the home pens were indelibly marked and distances between them measured. Every time participants passed a landmark, they were timed with a stopwatch until they passed another landmark. Time and landmarks were recorded, and walking speed was calculated as distance/time. Instances when animals moved away after being supplanted or chased, fled, or moved toward a food source or other desirable object, such as a toy, were not recorded. Walking bouts also had to be at least 1 minute. Each animal was observed until at least five instances of usual walking speed were recorded. To minimize the potential influence of circadian rhythms, observations were made over the course of several days, prior to morning feeding (06:00–09:00), when the monkeys were most active (26).

Climbing

Participants were observed for four 15-minute focal periods (ie, 1h/monkey) balanced for time of day (27). Each observation was done on a different day. The frequency and duration of climbing were recorded. Climbing, expressed as frequency, was defined as movements in which at least 2 feet were off the ground.

Time Locomoting and Leaping/Jumping

Locomotion was defined as traversing space at any speed with a minimum of three continuous steps. Leaping and jumping were recorded together (leaping/jumping) and defined as no body parts touching a surface (26).

Skinned Muscle Fiber Preparation

Experiments were performed in the VL, the muscle most widely used to examine the mechanisms underlying loss in force and composition in the aging human population. At the time of necropsy, the animals were deeply anesthetized with intravenous pentobarbital (60mg/kg). After exsanguination and a 5–10 minute flush with lactated ringer, muscle bundles were carefully dissected and immediately placed in cold (4°C) relaxing solution, where they were later divided into bundles of approximately 30–50 fibers and tied with 10-0 silk surgical suture to glass capillary tubes at slightly stretched lengths. The fiber bundles were chemically skinned for 24 hours in 50% relaxing solution and 50% glycerol at 4°C and subsequently stored at −20°C for up to 4 weeks.

Solutions

The composition of the relaxing and activating solutions used for single-fiber experiments were derived from the computer program described by Fabiato (28). Stability constants used in the calculations were adjusted for the total ionic strength (180 mmol), temperature (15°C), and pH (7.0) of the experiments (29). All relaxing and activating solutions contained 7.0mM ethylene glycol tetraacetic acid, 14.5mM creatine phosphate, 20.0mM imidazole, 4mM Mg²⁺–ATP, and 1mM free Mg²⁺. CaCl₂ was used to adjust the free Ca²⁺ concentration of the relaxing and activating solution to pCa 9.0 and pCa 4.5, respectively (where pCa = −log[Ca²⁺]). For both solutions, pH was adjusted to 7.0 with KOH and total ionic strength to 180mM with KCl.

Experimental Setup

A single-fiber segment was carefully isolated from a bundle using fine forceps. Both ends were securely tied to titanium wires, where one wire connector extended from an isometric force transducer (Model 403, Aurora Scientific, Aurora, Ontario) and the other connector was attached to a high-speed servomotor (Model 315C, Aurora Scientific) using 10-0 sutures. The length of the fiber suspended between the wires was about ~1.7±0.03mm. A 3-mm long wire connector extending from the motor was used to minimize any error in fiber length (FL) due to the rotation of the lever arm (30). The mounted fiber segment was suspended in one of several small glass-bottomed chambers formed in a stainless steel plate and activated by rapid motorized transfer of the stainless steel plate from the chamber containing relaxing solution to the chamber containing activation solution. The experimental apparatus was mounted on the stage of an inverted microscope (Axiovert S100, Carl Zeiss Inc., Germany), so we could observe the fiber at 450× through the transparent bottom of each chamber. Images of the fiber were obtained using a charge-coupled device camera and a scientific graphic acquisition board.

FL and sarcomere length were measured using a calibrated eyepiece micrometer set to ~2.5 µm by adjusting the overall segment length. Final sarcomere length was confirmed by measuring 10 consecutive sarcomeres on at least three places along the fiber. Fiber CSA was modeled as an ellipse and calculated from measurements of fiber width and depth by a calibrated eyepiece micrometer. Fiber width was measured at three points by recording the vertical displacement of the microscope nosepiece while focusing on the top and bottom surfaces of the fiber.

Single-Fiber Physiology Tests and Experimental Protocols

All measurements were conducted at 15°C, and temperature was continuously monitored by a thermocouple inserted into the experimental chamber. All functional data were collected and analyzed using a personal computer and a data acquisition board (Model 600A Digital controller, Aurora Scientific, Aurora, Ontario). A slack test was used to determine the unloaded shortening velocity (V _o) as described in Figure 1A. In this procedure, fibers were transferred to activating solution (pCa 4.5) and once peak force was attained (monitored by real-time digital oscilloscope), subjected to a rapid slack step (≤20% of FL within 1ms). The procedure was repeated at different slack lengths, and the times required for tension redevelopment were plotted versus the corresponding slack distances. A straight line was fit by least-squares linear regression, and the slope of the regression line, normalized to FL, defined V _o.

Figure 1.

Experimental protocol. (A) Superimposed length steps and the corresponding superimposed force records during a slack test. After reaching maximal fiber force, slacks of various amplitudes were rapidly introduced (upper panel). Rapid drop in force was followed by force redevelopment as the fiber was slacked (lower panel). The time for force redevelopment was plotted against the slack length step and fit by linear regression to yield V _o (per fiber length [FL]). Vertical bars represent 200 µm for length and 0.2 mN for force, respectively. The horizontal bar represents 100ms. (B) Superimposed fiber force records and the corresponding superimposed change in FL during three consecutive isotonic load clamps followed by a slack step to zero the transducer. Three force steps (isotonic contractions) were administrated for each activation. Isotonic force and the corresponding shortening velocity were calculated over the final half of each isotonic load clamp. Data were fitted by the Hill equation (32), (P + a)(V + b) = (P _o + a)b, where P is force, a and b are constants of force and velocity, respectively, V is velocity, and P _o is maximal force. Five to six series of three isotonic contractions were used to establish a force–velocity relationship (15–18 data points). Vertical bars represent 200 µm for length and 0.2 mN for force, respectively. The horizontal bar represents 100ms. (C) Representative silver stained 6% polyacrylamide gel illustrating type I, IIa, IIx and IIb myosin heavy-chain (MHC) isoform in skinned fiber segments, prepared from vervet monkey vastus lateralis (VL) muscle. Each lane contains a single muscle fiber segment. Lane 1: type I/IIa/IIx fiber; lane 2: type IIa/IIb; lane 3: type IIa.

After the slack test, the force–velocity relationship was generated by performing a series of isotonic contractions of the muscle fiber (Figure 1B; 31). Briefly, the muscle fiber was placed in activating solution (pCa 4.5) and after reaching peak force, subjected to a series of three isotonic steps varying from 3% to 80% of P _o. After the last step, the fiber was rapidly (<1ms) slackened by 20% of its length, which zeroed the force transducer, providing a baseline for force measurement. Step duration was less than ~100ms. Shortening velocity and force were obtained as averages over the final half of each step. Velocity was calculated as the slope of the position recorded over the same time period. Five to six series of three isotonic contractions were used to establish a force–velocity relationship.

The Hill equation was fitted to the data (32) using an iterative nonlinear curve-fitting procedure to draw the force–power relationship. The following parameters were used to describe the hyperbolic fit to the data: V _max (the velocity extrapolated to a force of zero), P _o (average force obtained during the trial), and a/P _o (a parameter describing the curvature or shape of the force–velocity relationship; 33). V _max was normalized to FL. a/P _o is a dimensionless parameter. Peak power was calculated from these three parameters, and expressed as W/L fiber (34). In all contractions, Ca²⁺-activated force was measured using the transducer zero signal as a baseline. Forces were normalized to the fiber’s CSA to obtain specific force.

Quality Control

Fibers were excluded from analysis if force declined more than 5% or if they broke or showed partial myofibrillar tearing at any observation timepoint in the experimental protocol. Experiments were excluded from analysis if compliance, defined as displaced axis-intercept of the slack test plot, exceeded 5% of FL (Table 2) and if the r ² of the force–velocity regression was less than .98. Average r ² values were .988 ± .001 and .987 ± .001 for young and old fibers, respectively.

Table 2.

Morphological and Functional Characteristics of Single Muscle Fibers

							Force
	MHC	No. of Fibers (%)	FL (mm)	CSA (µm2)	V 0 (FL/s)	Compliance (% FL)	(mN)	(kN/m2)
Y	IIa	51 (78)	1.7±0.0	7860±260*	5.63±0.26	3.2±0.2	0.94±0.03*	120±2*
	Hybrid	14 (22)	1.7±0.1	6720±550†	5.75±0.61	3.6±0.5	0.81±0.08*	120±4*
O	IIa	54 (76)	1.7±0.0	6120±240	4.95±0.22	3.1±0.2	0.63±0.03	102±2
	Hybrid	17 (24)	1.8±0.1	5940±450	4.43±0.49	3.4±0.3	0.63±0.05	107±3

Notes: Values are mean ± SE. CSA = fiber cross-sectional area; FL = fiber length; MHC = myosin heavy chain isoform; No. of fibers = number of VL muscle fibers from four monkeys for each age group; O = old monkeys; V _o = unloaded shortening velocity; Y = young monkeys. *—indicates significant difference between age groups (p < .05). †—indicates a significantly difference within age groups (p < .05), analyzed by two-way analysis of variance based on number of single fibers.

Assessment of Fiber Myosin Heavy-chain Isoform

At the end of each functional experiment, the single-fiber segment was removed from the test apparatus and stored in 20 µL of sodium dodecyl sulfate sample buffer (containing 62.5mM Tris pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 5% betamercaptoethanol, and 0.001% bromophenol blue) at −80°C. Later, fibers were denatured for 5 minutes at 95°C. To determine the myosin heavy-chain (MHC) composition of the fiber segment, a sample of the fiber solute, equivalent to that tested for contraction, was run on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis system that consisted of a 6% separating gel and a 4% stacking gel (acrylamide:bisacrylamide = 37:1; Figure 1C). The gels contained 30% glycerol to improve separation of MHC isoforms. Electrophoresis was carried out at 140V for 6 hours on a Bio-Rad mini-protean tetra electrophoresis system (Bio-Rad Laboratories, Hercules, CA). Silver staining was used to visualize the protein bands. Fiber MHC isoform content was compared with monkey MHC standards prepared from VL muscle samples run on one lane of each gel. This technique allows us to identify the type II MHC, whereas histology distinguishes type I from II fibers.

Histological Assessment of Muscle Fiber Type

Muscle samples were embedded in Optimal Cutting Temperature medium (Tissue-Tek, Torrance, CA), rapidly frozen in liquid nitrogen, and kept at −80°C for subsequent use. Sections (12 µm) were mounted on glass slides for ATPase staining as previously described (35). Briefly, slides were incubated in a 0.01M ATP solution containing 0.1M glycine/NaCl with 0.75M CaCl₂, pH 9.6 at 37°C for 15 minutes and then incubated in 2% COCl₂ for 5 minutes. The section was developed in ammonium sulfide solution (1:50) for 30 seconds. Slides were mounted on glycerin jelly. Type I and II muscle fibers were imaged using TSView 7.1 (Tucsen Imaging Technology, NY).

Statistical Analysis

SigmaPlot 11.0 (Systat Software, San José, CA) and SAS Proc mixed (SAS Inc., Cary, NC) were used for all statistical analyses. All data are presented as mean ± SE. As repeated measurements were made within subject, a mixed effects model was used to analyze the data (36). It was a generalization of analysis of variance and took into account the intramonkey clustering of the measurements. Briefly, the procedure first included MHC isoform, age group, and MHC × age as fixed effects and monkey as a random effect. If the interaction term was not significant, it was removed, and only the main effects of MHC and age were included in the model. The alpha level was set at p = .05 and all reported p values are the result of two-sided tests. Linear regression analysis was further used to identify the relationship between fiber CSA or physical performance tests and force. When reporting correlations between variables, we distinguished within-monkey from between-participants (37,38); correlations reported here were between-monkey, based on the means of the specific variables across fiber samples. In general, the simple Pearson correlation between-participant means of the repeated measures was known to approximate the true overall correlation between variables (36).

Results

Physical Performance Declines With Aging

We examined spontaneous walking speed, time spent locomoting, and incidence rate for leaping, jumping, hanging, and climbing in all monkeys whose muscles were tested in vitro. Old monkeys walked slower (19%) and climbed less (63%) than young monkeys (p < .05), but they did not exhibit any significant difference in the remaining tests (Table 1). Lower spontaneous physical performance measurements were not due to such dissimilarities in body composition as weight, body mass index, and percent of fat between young and old monkeys (Table 1).

Table 1.

Body Composition and Physical Performance Measurements

	Age (years)	Body Weight (kg)	BMI (kg/m2)	Fat (%)	Walk Speed (cm/s)	RT Climb	RT Leaping	RT Locomoting	RT EHanging
Y	11±1	5.6±0.6	60±4	27±3	58±3	12.0±1.1	8.0±1.2	7.7±0.5	2.3±0.5
O	23±1*	5.3±0.3	58±5	26±5	47±3*	4.5±2.6*	6.8±3.0	8.6±2.0	0.8±0.8

Notes: Values are mean ± SE. BMI = body mass index; RT = frequency/h; O = old monkeys; Y = young monkeys. *—indicates a significant difference between age groups (p < .05) analyzed by Student’s t test for unpaired data based on number of single fibers.

Fiber Length, Unloaded Shortening Velocity, and Compliance

We measured morphological and functional characteristics of 65 fibers from young and 71 from old monkeys (Table 2). No statistical difference in FL and compliance, defined as the displacement intercept of the slack test plots expressed as a percent of FL, were observed across ages and fiber subtype. Maximal unloaded shortening velocity (V _o), assessed by the slack test, also showed no statistical difference between age groups. However, fibers from old monkeys showed slower V _o for type IIa (~13%) and hybrid (~22%) fibers than fibers from young monkeys (Table 2). All fibers tested in this study were fast (type-II) or hybrid; we found no fibers expressing MHC type-I exclusively.

Fiber Type Composition

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed that 51 (79%) and 54 (76%) fibers from young and old monkeys, respectively, were type IIa. The remaining fibers, I/IIa, I/IIa/IIb, IIa/IIb, IIa/IIx, and IIa/IIx/IIb, were represented in the following quantities: 1 (1.5%), 4 (6.0%), 1 (1.5%), 4 (6.0%), and 4 (6.0%) in young and 1 (1.4%), 2 (2.8%), 7 (9.9%), 5 (7%), and 2 (2.9%) in old monkeys, respectively.

Due to the lack of slow myofibers in these experiments, we analyzed fiber type composition in ATPase (pH 9.6)–stained muscle cross-sections. Representative images showed few type-I fibers in young and old monkeys (Figure 2A). Analysis of 1,026 and 1,314 fibers from 3 young and 3 old monkeys, respectively, showed that 94% and 95% were fast, supporting the conclusion that the VL muscle of vervet monkeys consists mainly of fast fibers and that fiber type composition does not change with aging (Figure 2B).

Figure 2.

Muscle fiber type composition. A. Representative vastus lateralis muscle cross sections from young (A) and old (B) African Green vervet monkeys at low magnification. Enlarged regions of interest drawn in A and B are represented in A’ and B’, for young and old monkeys, respectively. Type-I slow-twitch fibers correspond to the light fibers (I) while type-II fast-twitch fibers are dark. B. Percent of type-I and type-II fibers in the vastus lateralis muscle from young and old monkeys. Values are mean ± SEM. Notice the muscle fiber atrophy with aging. * indicates a significant difference vs. corresponding fiber type in young monkey (p < 0.05)

Fast Fibers Atrophy With Aging

Fibers from old monkeys expressing MHC IIa isoform had a ~22% smaller CSA than those from young monkeys (p < .001). Fibers coexpressing either two or three isoforms showed about 15% smaller CSA than fibers containing a single MHC isoform in young (p < .05) but not old monkeys. Thus, aging results in decreased CSA in type IIa but not hybrid fibers. The CSA histogram of all fibers shows that while 59% of young monkey fibers are in the 7,000–10,000 µm² range, 60% of old monkey fibers are between 5,000 and 8,000 µm² (Figure 3A).

Figure 3.

Relationship between fiber cross-sectional area (CSA) and force. A. Histogram. The dark gray and light gray bars represent young and old monkeys, respectively. B. Scatterplot of fiber CSA and maximal Ca2+-activated force. Red diamonds and dashed regression line represent results from young monkey fibers. Light gray circles and solid regression line represent data from old monkey fibers. The linear regression analysis shows impaired force-generating capacity in old monkey fibers.

For the histological analysis of fiber CSA, we randomly measured 404 fibers (54 young slow, 150 young fast, 50 old slow, and 150 old fast fibers from 3 young and 3 old monkeys). Differences were significant for fast-twitch fibers (p < .01) but not for slow-twitch fibers, suggesting that predominantly fast fibers undergo atrophy with aging (Figure 2B). Notice the difference in CSA measured in isolated fibers and by histology; single muscle fibers swell 20% from the chemical skinning procedure (39,40). Fiber CSA measured in histological sections confirmed the age-dependent atrophy in single muscle fibers.

Maximal Contraction Force Decreases With Aging and Is More Pronounced in Fibers With Larger Diameter

Maximal type IIa fiber Ca²⁺-activated force (mN) and specific force (normalized to CSA, kN/m²) decreased by 33% and 15%, respectively, in old compared with young monkeys (p < .001). Likewise, the forces for fibers co-expressing multiple MHCs decreased by 22% (mN) and 11% (kN/m²) with aging (p < .05). Linear regression between fiber CSA and force (mN) showed a strong linear relationship for both young (r ² = .84, p < .001) and old (r ² = .82, p < .001) monkeys (Figure 3B).

Fiber Power Declines With Aging

Absolute power (µN × FL/s) recorded in type IIa fibers from old monkeys was substantially lower than in fibers from young monkeys (~40%, p < .001; Table 3). This loss in fiber power may be explained by a 33% reduction in Ca²⁺-activated force (mN) coupled to an 8% decrease in V _max since a/P _o did not differ. However, this substantial loss in power output was somewhat less obvious when absolute power was normalized by fiber volume (~21%, p < .001), which can be explained by a ~22% decrease in fiber CSA. Likewise, hybrid fibers from old monkeys showed a ~35% lower absolute power (µN × FL/s) than young monkeys (p < .05). The normalized power (W/L) also decreased to ~25% with aging (p < .05). We did not detect any statistically significant difference in a/P _o or V _max across age groups per specific fiber type.

Table 3.

Myofiber Force–Velocity Relationship

					Peak Power
	MHC	No. of fibers	V max (FL/s)	a/P o	µN × FL/s	W/L
Y	IIa	44	4.89±0.20	0.039±0.00	119±9	14.5±0.5
	Hybrid	11	5.29±0.74	0.040±0.00	109±15	15.0±1.3
O	IIa	47	4.52±0.22	0.039±0.00	71±4*	11.5±0.4*
	Hybrid	14	4.42±0.55	0.037±0.00	71±11*	11.2±1.3*

Notes: Values are mean ± SE. MHC = myosin heavy chain isoform; No. of fibers = number of fibers obtained from vastus lateralis muscle from four monkeys for each age group; O = old monkeys; Y = young monkeys; V _max = y-intercept of force–velocity relationship; a/P _o = unitless parameter describing the curvature of the relationship. *—indicates a significant difference between age groups (p < .05) analyzed by two-way analysis of variance based on number of single fibers.

Age-dependent Decrease in Myofiber Force and Physical Performance

A linear regression analysis was used to examine the relationship between physical performance measurements and single-fiber contractile properties. A strong negative relationship (r = −.927, p < .001) was observed between a monkey’s age and specific force (kN/m²; Table 4). A positive relationship was revealed between walking speed and maximal fiber-specific force (r = .64; Table 4). Likewise, there was positive relationship between the monkey’s climbing rate and specific force (r = .469); however, the correlation between these tasks and fiber-specific force is not statistically significant. Leaping/jumping, hanging, and locomotion correlated weakly with specific force.

Table 4.

Relationship Between Physical Performance Measurements and Single-Fiber Contractile Properties

		CSA	Force	Power
		µm2	kN/m2	W/L
Age	r	−0.860	−0.927	−0.855
	p	.006	.000	.007
RT time climb	r	0.420	0.469	0.609
	p	.300	.242	.109
RT time leaping	r	0.180	0.183	0.367
	p	.670	.665	.371
Percent time locomoting	r	−0.406	−0.399	−0.120
	p	.318	.327	.777
Mean walking speed	r	0.498	0.640	0.594
	p	.209	.088	.121
RT hang	r	0.559	0.320	0.282
	p	.150	.439	.499

Notes: CSA = fiber cross-sectional area; RT = relative to time; r = Pearson correlation coefficient.

Discussion

The goal of this study was to investigate the effect of aging on locomotion and intrinsic myofilament contractile properties in young and old monkeys under controlled environmental and nutritional conditions. The physical performance measurements revealed that old monkeys walked slower and climbed lesser than young monkeys. The functional assay of muscle fibers showed a smaller fiber CSA and lower force and power-generating ability of fast and hybrid fibers isolated from the VL muscle of old compared with young monkeys. Unloaded shortening velocity was independent of age, regardless of the fiber type.

Previous Studies on Human Muscle Fiber Function Show Contradictory Results

Despite previous efforts to elucidate the effect of aging on intrinsic myofilament contractile properties, such as maximal shortening velocity and specific force in human myofibers, the exact relationships remain uncertain (23). Current literature on cross-sectional studies, compiled in Table 5, reflects such inconsistencies in human muscle. No comparable studies in aging nonhuman primates have been reported. While 2 out of 10 studies found significantly smaller CSA in the type I fibers, the remaining studies reported no difference with aging. Six studies concluded that type IIa fibers exhibit a smaller CSA in the older monkeys, whereas seven studies found no difference. Overall, these studies suggest that type I fiber size may be well maintained with aging, whereas type IIa fiber size may have a tendency to decline, but neither is certain due to contradictory information. A comparable number of studies show either preserved or decreased specific force or maximal shortening velocity in type I and IIa fibers with aging. On the other hand, only four studies measured power output using the force–velocity relationship. While three studies reported maintained or even slightly increased power in both fiber types, only one found lower power output in both. These contradictory findings on intrinsic cross-bridge kinetics may derive from the wide variability of subject characteristics, particularly physical activity levels, as pointed out previously (24).

Table 5.

Summary of Published Data on Fiber csa and Contractile Properties of Human VL Muscle Fibers

Age (N)					CSA		Normalized Force		Velocity		Power
Young	Old	Activity Level	Sex	Muscle	I	IIa	I	IIa	I	IIa	I	IIa	References
30±2 (7)	73±2 (7)	Inactive	M	VL	−22%	−12%	−22%	−16%	↓	↓	N/A	N/A	11
32 (6)	66 (6)	Inactive	M	VL	=	=	−25%	−33%	−23%	−22%	N/A	N/A	13
37 (7)	74 (12)	Inactive	M	VL	=	=	−28%	−31%	N/A	N/A	N/A	N/A	14
20−43 (7)	65–85 (17)	N/A	M	VL	=	−29%	−23%	−14%	−17%	−11%	N/A	N/A	59
20−43 (6)	65–85 (5)		F		=	=	−20%	−25%	−20%	−30%	N/A	N/A
25−31 (4)	73–81 (2)	Combined	M	VL	=	−28%	=	−28%	=	=	N/A	N/A	16
25±3 (33)	76±4 (30)	Inactive	N/A	VL	11%	−24%	=	=	↓	=	↓	↓	60
37±3 (7)	74±6 (7)	Inactive	M	VL	N/A	/A	N/A	N/A	=	−17%	N/A	N/A	15
27±3 (12)	72±4 (12)		F		N/A	N/A	N/A	N/A	−7%	=	N/A	N/A
30±4 (5)	73±3 (3)	Inactive	N/A	VL	N/A	N/A	↓	↓	↓	↓	N/A	N/A	24
	73±3 (3)	Trained	N/A		N/A	N/A	=	=	=	=	N/A	N/A
21±2 (9)	85±1 (6)	Inactive	F	VL	=	=	=	=	=	=	=	=	21
25±1 (6)	80±4 (6)	Inactive	M	VL	=	=	=	12%	=	=	=	=	20
25±1 (6)	78±2 (6)		F		13%	−17%	=	=	=	=	=	=
18−33 (8)	53−77 (9)	Sprinter	M	VL	−26%	−36%	=	=	−24%	=	N/A	N/A	18
23±1 (9)	67±2 (8)	Inactive	M	VL	=	=	=	=	N/A	N/A	N/A	N/A	50
27±3 (7)	74±4 (7)	Inactive	F	VL	=	=	=	=	N/A	N/A	N/A	N/A	19

Notes: Values represent percent difference between young and old participants; CSA = fiber cross-sectional area; F = female; M = male; N = number of participants; N/A = not available data; VL = vastus lateralis; =, no statistical difference between young and old; ↓ = reported significant decrease in old compared with young but data not provided;

In this work, we found a smaller fiber CSA and lower specific force and power output in type II fibers of old monkeys. A multiple regression analysis revealed age as the strongest predictor of fibers’ functional capacity; that is, maximal force (kN/m², r ² = .86, p < .001) and power (W/L, r ² = .73, p < .001). Therefore, a single muscle fiber’s intrinsic capacity to sustain force and power appears to degrade with aging. Surprisingly, all fibers dissected and tested here expressed either IIa MHC or multiple MHC isoforms but not type I alone. A histological analysis of fiber-type composition confirmed the small percentage of type I fibers in VL muscle from vervet monkeys. Our results are limited to fast fibers (IIa MHC) and fibers co-expressing two or three MHC isoforms. We did not observe any fiber that expressed only slow type I MHC isoform.

Adult mammal limb muscles express four different MHC isoforms, type I, IIa, IIx, and IIb (41), though type IIb isoform is not found in the limb muscles of large mammals, including humans (42,43). Very little information is available regarding nonhuman primate muscle MHC expression patterns. Three MHC isoforms, type I, IIa, and IIx, were identified in the soleus and medial gastrocnemius muscle from rhesus monkeys (44,45); type IIb MHC was also found in VL muscle in rhesus (46) and squirrel monkeys (47). The present study shows that VL from vervet monkeys consist predominately of fast muscle fibers, including type IIb MHC, which is consistent with a previous study reporting more than 75% of the fibers of the fast MHC isoform, including IIb MHC in the VL muscle of the rhesus monkey (46).

Contractile properties of single fibers from lower limb skeletal muscle obtained from nonhuman primates more resembled those of humans than small rodents (45,48). The specific force recorded in this study ranged from 67 to 138kN/m², in good agreement with previous studies conducted under similar experimental conditions in humans (21,22,49,50). Unloaded shortening velocity (V _o) was inversely related to species body mass, as previously observed in humans, monkeys, rats, and horses (48,51). Vervet VL Vo was approximately 30% higher than humans’ (20,21) and 20% lower than small rodents’ (52).

These interspecies differences might be explained by inherent differences in cross-bridge kinetics. However, the natural history of nonhuman primate aging has not been well defined in relation to humans. As indicated in Table 5, studies on older human single muscle fibers reported decreased morphological and contractile properties, and a 12%–30% smaller diameter compared with fibers from young participants. The present study found a moderate decline (~22%) in type IIa fiber diameter. Hybrid fibers from old monkeys were about 12% smaller in diameter, but this difference is moderate compared with type IIa fibers. In humans, maximal Ca²⁺-activated specific force declined from 14% to 33% with aging in type IIa fibers; 15% is consistent with the lower end in human studies. Although, we found slower V _o in old monkeys, the difference was not statistically significant when compared within fiber types due to wide variation (1.87 ~ 10.26 FL/s). Consequently, the morphological and contractile properties and adaptation to aging of single muscle fibers prepared from nonhuman primates strongly resembles the human preparation.

Skeletal Muscle Contractility Declines With Aging in African Green Vervet Monkeys

Gait speed predicts disability, hospitalization, and mortality in humans (53–55). In this study, we quantified monkeys’ spontaneous activity as maximal performance requires more invasive procedures. We recently reported (26) that walking speed is the most sensitive physical performance test of age in three nonhuman primate species. Here, linear regression analysis shows that walking speed and single muscle fiber-specific force tend to associate (r = .64, p < .088). Lower force-generating capacity of single muscle fibers explains 41% of variation in walking speed (r ² = .41). A walking speed of 5cm/s decreases fiber force to 12kN/m². Further studies with larger samples are needed to pursue these structure–function relationships.

The present study revealed that absolute force impairment increases with fiber CSA in old monkeys. The mechanism accounting for large fibers’ deviation from classical force–CSA behavior is unknown, but two mechanisms have been proposed: fewer strongly bound actin–myosin cross-bridges during maximal activation (12) and/or a reduced ability of each cross-bridge to generate force (13). According to Lowe and colleagues (12), when muscle fiber contracts maximally, about 32% of myosin heads are in a strong binding state, and only 22% are in that state in fibers from older rats. A strong correlation between myosin concentration and force has been reported, supporting the concept that a main cause for decreased P _o in older men is a decrease in the number of contractile myofibrils per CSA (11). Slower shortening velocity with aging is thought to involve slowing of the kinetic steps within cross-bridge cycling, such as the actin–myosin cross-bridge detachment rate (16). These functional alterations could occur without changes in fiber MHC isoform (56,57) and may be related to myosin glycation (58).

In summary, our results revealed that type IIa and hybrid fibers prepared from aged monkey VL muscle showed atrophy and reduced force and power output compared with young adult monkeys. This study supports a detrimental effect of aging on intrinsic contractile properties of VL myofilament lattice under controlled conditions. Impaired single muscle fiber mechanics was strongly correlated with the monkey’s age, tends to be associated with walking speed, and may implicate a causal link, which merits further investigation.

Funding

This work was supported by the National Institutes of Health grants (AG13934 and AG15820 to O.D., AG020583 to B.N.) and pilot and other funding from the Wake Forest University Pepper Older Americans Independence Center (P30-AG21332). The Vervet Research Colony was supported by a National Center for Research Resources grant (P40-RR019963).