Collagen, cross-linking, and advanced glycation end products in aging human skeletal muscle

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Collagen, cross-linking and advanced glycation endproducts in aging human skeletal muscle

Authors: Jacob M. Haus, John A. Carrithers, Scott W. Trappe, and Todd A. Trappe

Institutions: Human Performance Laboratory, Ball State University, Muncie, IN

Running Title: Intramuscular connective tissue and aging

Key Words: hydroxyproline, hydroxylysylpyridinoline, pentosidine, myosin, actin, sarcopenia

Address for correspondence: Todd Trappe, Ph. D. Human Performance Laboratory Ball State University Muncie, IN 47306 Ph: 765 285-4456 Fax: 765 285-8596 e-mail: [email protected]

Page 1 of 38Articles in PresS. J Appl Physiol (September 27, 2007). doi:10.1152/japplphysiol.00670.2007

Copyright © 2007 by the American Physiological Society.

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ABSTRACT

We examined intramuscular endomysial collagen, cross-linking and advanced glycation

endproducts, as well as the general and contractile protein concentration of 20 young

(25±3y) and 22 old (78±6y, range: 70-93y) sedentary men and women to better

understand the underlying basis of changes in skeletal muscle mass and function that

occur with aging. The old individuals had an impaired ability (increased time) (p<0.05)

to climb stairs (80%), rise from a chair (56%), and walk (44%), as well as lower (p<0.05)

quadriceps muscle volume (-29%), muscle strength (-35%), muscle power (-48%), and

strength (-17%) and power (-33%) normalized to muscle size. Vastus lateralis muscle

biopsies revealed that intramuscular endomysial collagen (Young: 9.6±1.1, Old:

10.2±1.2 µg•mg muscle wet wt-1) and collagen cross-linking (hydroxylysylpyridinoline,

HP) (Young: 395±65, Old: 351±45 mmol HP•mol collagen-1) were unchanged (p<0.05)

with aging. The advanced glycation endproduct, pentosidine, was increased (p<0.05)

by ~200% (Young: 5.2±1.3, Old: 15.9±4.5 mmol pentosidine•mol collagen-1) with aging.

While myofibrillar protein concentration was lower (-5%, p<0.05), the concentration of

the main contractile proteins myosin and actin were unchanged (p>0.05) with aging.

These data suggest that the synthesis and degradation of proteins responsible for the

generation (myosin and actin) and transfer (collagen and pyridinoline cross-links) of

muscle force are tightly regulated in aging muscle. Glycation related cross-linking of

intramuscular connective tissue may contribute to altered muscle force transmission

and muscle function with healthy aging.

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INTRODUCTION

A critical component in the transfer of force from the contractile units of the

muscle out to the tendon and subsequent bone is the connective tissue scaffold that

surrounds individual muscle fibers, muscle bundles, and the whole muscle (20, 23, 37).

This skeletal muscle connective tissue network is primarily comprised of collagen fibers

and biochemical linkages within and between these fibers, which provide strength and

stability. Numerous data from animals have shown that collagen concentration of intact

skeletal muscle increases with aging (1, 12, 17, 24, 25, 34, 55), and a mature structural

characteristic of collagen known as pyridinium cross-linking has also been shown to be

increased in aged animal muscle (17, 18, 36, 40, 55). The pyridinium cross-links are

enzymatically added to collagen fibrils upon collagen maturity and replace an immature

cross-link molecule. These mature pyridinium cross-links provide strength to the

collagen molecule by linking the collagen fibrils in the characteristic one-third staggered

arrangement.

Separate from the enzymatically derived, mature cross-links are an additional

form of biochemical linkage found on connective tissue known as advanced glycation

endproducts (AGE’s). Accumulation of these compounds are the result of prolonged

exposure to monosaccharides, in which a spontaneous non-enzymatic bond is formed

between a reducing sugar and a protein residue. AGE’s have been linked to disease

and aging processes (2, 10, 35, 38, 41) and have been demonstrated to be strongly

correlated to increases in plasma glucose concentrations and chronological age.

Evidence from humans and animals demonstrates the formation of these compounds in

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vascular tissues and lens proteins with diabetes and has shown a progressive

accumulation in skin and cartilage with aging.

Skeletal muscle studied in aging animals demonstrates increases in collagen

concentration, HP cross-linking, and AGE’s, all of which have been associated with

increased muscle stiffness and reduced whole muscle function (1, 12, 17, 26, 52).

Increases in tissue stiffness have been suggested to play a role in reduced whole

muscle function with aging. However, almost no data exist from humans regarding

intramuscular connective tissue changes with aging (3) or the extent of collagen cross-

linking or the development of AGE’s in healthy human skeletal muscle (14, 45). In

aging humans, muscle strength and power is lost to a greater extent than the loss of

muscle mass (i.e., whole muscle specific force and specific power), suggesting that

factors other than the loss of muscle mass are contributing to the decline in whole

muscle function (47). We have shown that single muscle fiber strength and power

normalized to fiber size [i.e., myosin heavy chain (MHC) I and IIa myofiber specific force

and specific power] are not compromised with aging (47). These findings lead to the

possibility that the transfer of force and power from the single fiber out to the whole

muscle and subsequent tendon and bone may be impaired in aging skeletal muscle.

The intent of the current investigation was to expand on our previous findings of

the role of single muscle fiber function in the decline of whole muscle function in aging

through measurements of the intramuscular connective tissue network. Specifically, we

measured the concentration of collagen (endomysium), collagen cross-linking

(hydroxylysylpyridinoline, HP), and AGE’s (pentosidine) in skeletal muscle biopsy

samples taken from a well-characterized group of young and old sedentary women and

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men. We hypothesized that the concentrations of endomysial collagen, HP cross-links

and pentosidine would be elevated in old individuals compared to young. An additional

aim was to measure the concentration of the general muscle protein fractions (mixed,

sarcoplasmic, and myofibrillar) commonly studied in metabolic investigations of

sarcopenia (5, 19, 42, 50, 51, 54), as well as the concentration of the two main

contractile proteins that are responsible for force generation in skeletal muscle, myosin

and actin. These measurements extend our previous investigation from a subset of the

42 individuals studied for the present investigation (47).

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METHODS

Subjects

Twenty young (10 men, 10 women) and 22 old (10 men, 12 women) individuals

were included in this investigation (Table 1) following a physical exam, which included

blood and urine analyses, electrocardiogram (older subjects), and an interview to

document their life history of physical activity. Subjects were excluded if they had any

acute or chronic illness, cardiac, pulmonary, liver, or kidney abnormalities, uncontrolled

hypertension, insulin- or noninsulin-dependent diabetes, abnormal blood or urine

chemistries, arthritis, a history of neuromuscular problems, or if they smoked tobacco.

Women taking oral contraceptives or hormone replacement therapy were included. It

was our intent to only include life-long sedentary healthy older and younger individuals;

therefore, we excluded individuals that, by self-report, had ever completed any formal

exercise programs or physical activity outside of their activities of daily living. All

procedures, risks and benefits associated with the experimental testing were explained

to the subjects prior to signing a consent form adhering to the guidelines of the

Institutional Review Boards of the participating institutions. The study was conducted in

accordance with the Declaration of Helsinki.

Experimental Design

Subjects who qualified for the study completed six different experimental sessions

over a two to three week period, described in detail below. The first session involved

magnetic resonance imaging (MRI) for thigh muscle size. The second session involved

the measurement of body composition via whole body air-displacement plethysmography

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(Life Measurement Instruments, Concord, CA) and a muscle biopsy of the vastus

lateralis. The remaining four visits involved the measurement of functional ability and

muscle function. The first two of these four visits were used for familiarization to the

tests, while the third was used for data analysis (i.e., the data presented here) and the

fourth for the determination of the reliability of these measurements. Each of these four

visits was separated by at least two days. The muscle biopsy and the measurements of

muscle size and strength were completed on the dominant leg. Dominance was

determined by asking the subject which leg they would use to kick a ball.

Magnetic Resonance Imaging

Following one hour of supine rest to control for the influence of postural related

fluid shifts on muscle size (8), MRI were obtained for each subject as we have previously

described (48). Subjects were supine and their heels were fixed on a non-metallic

support to control joint and scan angle and to minimize compression of the legs against

each other and the MRI gurney. Imaging was completed in a 1.5T GE Signa scanner

(General Electric, Milwaukee, WI) to determine the volume and cross sectional area

(CSA) of the total quadriceps femoris, rectus femoris (RF), vastus lateralis (VL), vastus

intermedius (VI), and vastus medialis (VM). Following an initial scout scan, interleaved

transaxial images of 1 cm thick (TR/TE = 2000/9.0 ms, field of view 48 cm, 256 X 256

matrix) were taken from the top of the greater trochanter of the femur to the articular

surface of the tibia. MR images were transferred electronically from the scanner to a

personal computer (Macintosh Power PC) and analyzed with NIH Image software

(version 1.60) using manual planimetry. Analyses of the MR images began with the first

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proximal slice not containing gluteal muscle and continued distally to the last slice

containing RF. The average CSA (cm2) was taken as the average of all the analyzed

slices for an individual muscle and determined for the RF, VL, VI, and VM and summed

for the total quadriceps femoris. Muscle volume (cm3) was calculated by multiplying the

CSA of each individual muscle by the slice thickness (1 cm) for all analyzed images of

the RF, VL, VI, VM, and summed for the total quadriceps femoris. All measurements

were made by the same investigator.

Muscle Biopsy

Each subject underwent a muscle biopsy (9) of the vastus lateralis for the

measurement of intramuscular connective tissue, general and contractile proteins and

MHC distribution. Following the biopsy, excess blood, visible fat and connective tissue

were removed and portions of the muscle to be used for the aforementioned analyses

were immediately frozen and stored in liquid nitrogen (-190°C) until analysis. An

additional portion of the muscle was used to study the single muscle fiber contractile

characteristics of a subset (n=24) of the subjects and the results of those experiments

are presented elsewhere (47).

Intramuscular Connective Tissue Quantification

Collagen concentration. Skeletal muscle collagen concentration was determined

via measurement of the collagen specific amino acid, hydroxyproline (HYP). The

concentration of HYP was quantified by high-performance liquid chromatography and

fluorometric detection (1100 Series, Agilent Technologies, Wilmington, DE, USA) via the

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precolumn derivatization method described by Hutson et al. (21) with modifications for

human skeletal muscle. Muscle samples (~10 mg) were weighed at -35°C on a

precision microbalance (AD-2Z Autobalance, Perkin-Elmer, Wellesley, MA, USA) and

hydrolyzed in 1 ml of 6 M HCl at 110°C for 30 h. In addition, a sarcosine internal

standard (Sigma #S7672, St. Louis, MO) prepared in water was added to each vial.

The hydrolyzates were allowed to cool to room temperature and neutralized with 6 M

NaOH. Hydroxyproline standards (Sigma #56250) of 1, 10, 25, 50, 75, 100, and 125

µM were prepared along with the 2 mM sarcosine internal standard, and were used to

generate a standard curve.

Derivatization was performed according to the procedures described by Hutson

et al. (21) and separation was achieved through a XTerra RP 18, 5 µm, 250 mm x 4.6

mm column (Waters, Milford, MA, USA) using an isocratic mobile phase of 65% acetic

acid (3% glacial acetic acid, sodium acetate buffered to pH 4.3): 35% acetonitrile at a

1.0 ml•min-1 flow rate. Peaks were monitored at 260/316 nm (Ex/Em) with a gain of 8

and integrated with chromatography software (ChemStation, Agilent Technologies)

(Figure 1). HYP signals were normalized to the internal standard for each injection and

HYP concentration was determined from standard curves of HYP (correlation coefficient

of 0.983). All samples and standards were run in triplicate with a mean coefficient of

variation of 0.32% and 0.69% respectively. Collagen concentration was calculated from

the HYP concentration assuming collagen weighs 7.5 times the measured HYP weight

and the molecular weight of collagen is 300,000 (13, 14). Samples were normalized to

muscle wet weight and data are expressed as µg collagen•mg wet weight muscle-1.

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Collagen cross-link concentration. The extent of collagen cross-linking was

determined by measuring the amount of the pyridinium cross-link

hydroxylysylpyridinoline (HP) through HPLC as previously described (6) with

modifications for human skeletal muscle. Muscle samples (~10 mg) were weighed at -

35°C and immediately placed into screw cap vials containing 1 ml of 6 M HCl after which

the samples were hydrolyzed at 110°C for 20 h. A 500 µl aliquot of the hydrolyzate was

then added to a sample cocktail consisting of a 4:1:1 ratio of butanol: cellulose slurry:

acetic acid containing acetylated pyridinoline (Quidel Corp. #8006, San Diego, CA) as an

internal standard.

Samples for collagen cross-links were extracted through CF1 cellulose partition

chromatography as described previously (43). The cross-links were eluted with water

and evaporated to dryness in a Savant Speedvac sample concentrator. Samples were

reconstituted in 0.5% heptafluorobutyric acid (HFBA) in 10% ACN and then filtered

(0.22 µM nylon filter) through centrifuge spin columns (Costar Spin X) at 10,000 x g for

10 min to remove residual cellulose particles.

HP standards (Quidel Corp. # 8004) of 5, 10, 25, 50, 75 and 100 pmol were

prepared in 0.5% HFBA/10% ACN buffer with acetylated pyridinoline added as an

internal standard. Standards and samples were then injected into the HPLC (Agilent

Technologies) and eluted as previously described with modifications (6). The column

(XTerra RP 18, 5 µm, 250 mm x 4.6 mm, Waters) was equilibrated with 0.13% HFBA in

22% methanol and cross-links were eluted with a 1.0 ml • min-1 flow rate from 0 - 30

min. A washing step followed immediately using 0.1% HFBA in 75% ACN for 10 min.

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Fluorescence of HP was monitored at 295/395 nm (Ex/Em) and integrated with

chromatography software (ChemStation, Agilent Technologies) (Figure 1).

Standard and sample signals were normalized to the internal standard signal and

sample HP recovery was then calculated as the percentage of sample internal standard

signal relative to the mean internal standard signal obtained from the known HP

standard curve. Mean recovery of skeletal muscle HP was ~ 80% after CF1 purification.

The concentration of skeletal muscle HP was determined from the standard curve and

adjusted for recovery. Cross-link concentration of each tissue sample was expressed

as mmol of HP per mol collagen assuming the molecular weight of collagen is 300,000

(7).

Advanced glycation endproduct (AGE) concentration. The AGE, pentosidine, was

measured in human skeletal muscle through HPLC and fluorometric detection (Agilent

Technologies). Muscle samples (~10 mg) were weighed at -35°C and immediately

placed into screw cap vials containing 1 ml of 6 M HCl. Samples were then hydrolyzed

at 110°C for 20 h. Following hydrolysis, samples were evaporated to dryness in a

Savant Speedvac sample concentrator followed by reconstitution with 0.5% HFBA/10%

ACN buffer containing 300 pmol of pyridoxine internal standard (Sigma #P5669). After

reconstitution the samples were filtered (0.22 µM nylon filter) through centrifuge spin

columns (Costar Spin X) at 10,000 x g for 10 min. Recovery of internal standard after

spin column filtration was tested and found to be 100%. Pentosidine standards of 0.05,

0.1, 0.25, 0.5, 0.75, 1.0, 2.5, and 5 pmol were prepared from a known concentration of

purified pentosidine obtained from the International Malliard Reaction Society (Vincent

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Monnier, CWRU, Cleveland, OH) in the same buffer as the samples (0.5%HFBA/10%

ACN), which included 300 pmol of pyridoxine.

The column (XTerra RP 18, 5 µm, 250 mm x 4.6 mm, Waters) was equilibrated

with 0.05% HFBA in water and the standards and samples were subsequently injected

into the HPLC (Agilent Technologies). Pyridoxine was eluted at ~ 10 min with an

inverse gradient of HFBA (0.05% to 0%; 0-20 min) and methanol (0% - 2.5%; 0-20 min)

at 1.0 ml • min-1 flow rate. Pentosidine was eluted at ~ 22 min with 2.5% methanol from

20-30 min. The elution gradients were followed by a washing step of 100% acetonitrile.

Fluorescence was monitored at 295/395 at a gain of 12 from 0-17 min for detection of

pyridoxine and 328/378 at a gain of 15 from 17-25 min for the detection of pentosidine

(Figure 1). Peaks were integrated with chromatography software (ChemStation, Agilent

Technologies) and standard and sample signals were reduced with and without

normalization to pyridoxine internal standard. Normalization had no effect on the

concentrations of pentosidine compared to raw signal when calculated from the

standard curve. Skeletal muscle pentosidine concentration was expressed as mmol per

mol collagen, assuming the molecular weight of collagen is 300,000 (7).

General Protein Quantification

Mixed, sarcoplasmic, and myofibrillar muscle protein concentration. For each

subject, a piece of muscle weighing ~10 mg was divided and weighed on a precision

microbalance (Cahn 35, Orion Research, Beverly, MA) at -35°C. Each sample was

homogenized in 40 volumes of cold homogenizing buffer (250 mM sucrose, 100 mM

potassium chloride, 20 mM imidazole, and 5 mM EDTA; pH 6.8) in a ground-glass

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homogenizer (Radnoti Glass Technology, Monrovia, CA) (11). Samples were then

centrifuged at 20,000 x g for 30 min at 4°C. The supernatant was taken as the

sarcoplasmic protein fraction and the pellet was resuspended in 40 volumes of cold

homogenizing buffer and taken as the myofibrillar protein fraction (22, 44). Aliquots of

the homogenate (total mixed protein), sarcoplasmic, and myofibrillar protein fractions

were measured for protein concentration using the bicinchoninic acid (BCA) assay

(Sigma) with bovine serum albumin used as the protein standard. The amount of protein

in each of the three fractions was normalized to muscle wet weight and expressed as µg

protein•mg wet weight muscle-1.

As collagen is contained in the mixed protein and myofibrillar protein (i.e. collagen

pellets following centrifugation) (3, 32, 33), follow-up BCA protein assays with pure

collagen (Sigma #C9791) were completed to confirm that collagen contributes to the

overall absorbance of the muscle measurements (Figure 2). These measurements were

completed because it has been suggested that collagen does not contribute to some

assays of mixed protein in skeletal muscle (39, 53).

Contractile Protein Quantification

Myosin and actin concentration. Myosin (myosin heavy chain, MHC) and actin

concentrations were determined by quantitative gel electrophoresis as previously

described (22, 49). Aliquots of the myofibrillar protein fraction were diluted with sodium

dodecyl sulphate (SDS) buffer (2% SDS, 125 mM Tris HCl (pH 6.8), 12.5% glycerol, 5%

2-mercaptoethanol, 0.005% bromophenol blue) and heated at 60°C for 4 min.

Myofibrillar protein (800 ng) was separated by SDS-polyacrylamide gel electrophoresis

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(PAGE) (28). MHC was resolved with a 4% stacking gel and 10% separating gel. Actin

was resolved with a 4% stacking gel and a 6-12% gradient separating gel that were

allowed to polymerize overnight. For MHC and actin, electrophoresis was performed at

a constant current of 20 mA per gel in the stacking gel, and 25 mA per gel in the

separating gel with a Tris-glycine electrode buffer at 4°C (Hoeffer SE 600, Amersham

Pharmacia Biotech, Piscataway, NJ).

The separating gels were silver stained (15), digitally photographed (ChemImager

5500, Alpha Innotech, San Leandro, CA), and densitometry was completed using NIH

Image software (Ver. 1.60) (47). Each gel was loaded with five standards of either MHC

or actin (Sigma #’s M7659, A2522), a molecular weight standard (Pierce, Rockford, IL),

and subject samples. All standards and samples were loaded in duplicate and each gel

contained samples from young and old men and women. An average of the duplicate

densities was taken to represent each standard and sample. All measurements were

made in blinded fashion by the same investigator. Unknown sample amounts of MHC

and actin were determined from regression analysis of the standard curves on each gel.

Correlation coefficients were 0.98-1.00 for both MHC and actin. The amount of myosin

or actin was normalized to muscle wet weight and expressed as µg•mg wet weight

muscle-1.

MHC Isoform Distribution

MHC isoform composition of each of the myofibrillar protein samples quantified for

myosin and actin protein concentrations was determined in triplicate from a SDS buffer-

diluted aliquot of the myofibrillar protein fraction heated to 100°C for 5 min. MHC

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isoforms were separated with SDS-PAGE with a 3.5% stacking gel and 5% separating

gel. Electrophoresis was performed at 150 V for ~15 h with a Tris-glycine electrode

buffer at 4°C (Hoeffer SE 600, Amersham Pharmacia Biotech, Piscataway, NJ). The

separating gels were silver stained (15), digitally photographed (ChemImager 5500,

Alpha Innotech, San Leandro, CA), and densitometry was completed to determine the

percent contribution of each isoform of the total (100%). The average density of each

isoform from the three lanes loaded was taken as the MHC distribution for that sample.

Functional Ability

The ability to perform activities of daily living was determined with three

functional tests: walk, chair stand, and stair climb. Each of the functional tests was

completed three times during each session, with the average of the three tests

recorded. Timing of the functional tests was accomplished using retroreflective sensors

(Banner Engineering Corp., Minneapolis, MN) interfaced to a digital timer and display

(Veeder-Root, Danaher Controls, Gurnee, IL). The sensors were placed such that the

forehead (chair stand or walk tests) or foot (stair climb test) would break the light beams

to start and stop timing.

Walk test. Subjects walked (i.e., one foot in contact with the ground at all times)

a distance of 6.1 m as quickly as possible. Subjects started three steps behind the first

photo eye and continued through the second photo eye. The coefficient of variation for

the walk test was 3.8 ± 0.5% with no group, age, or gender differences.

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Chair stand test. Subjects stood from a seated position in a metal chair as fast

as possible without support (i.e., via chair or thighs). The coefficient of variation for the

chair stand was 6.9 ± 0.9% with no group, age, or gender differences.

Stair climb test. Subjects started two steps before the first stair and climbed a

flight of 10 stairs (3.3m vertical distance) as quickly as possible without skipping a stair.

The coefficient of variation for the stair climb was 3.6 ± 0.5% with no group, age, or

gender differences.

Muscle Function

Following the functional ability measurements, subjects completed light

stretching of the legs and a five-minute warm-up on a bicycle ergometer (Monark

Exercise AB, Sweden). Muscle function testing was performed on a Cybex Norm

dynamometer (Lumenex, Ronkonkoma, NY) and associated equipment fabricated by

the investigators. Measurements were made of in-vivo maximal shortening velocity

(Vmax), as well as static and dynamic muscle strength and power of the quadriceps

femoris muscle. Adequate rest periods separated all of the tests within each testing

session.

Maximal shortening velocity. Vmax was determined by having each subject

extend their lower leg from a metal bar placed behind their heel to establish a starting

position at 90° of knee flexion through a 90° arc and into a heavily padded bar as fast as

possible. Fiber optic sensors (Banner Engineering Corp., Minneapolis, MN) interfaced

with a computer timer (LabVIEW, National Instruments, Inc., Austin, TX) were

positioned at 60° and 30° of knee flexion to measure the time to move through the 30°.

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Subjects were allowed three warm-up attempts followed by three timed leg extensions,

with the fastest time of the three recorded. The coefficient of variation was 3.5 ±0.5%

with no group, age or gender differences.

Static and dynamic force and power. Following completion of the Vmax

measurements, maximal isometric (Po), and concentric muscle forces were determined.

Po was measured at 60° of knee flexion and subjects were allowed one warm-up trial

followed by two maximal contractions, with the greater value taken as Po. A one-minute

rest period separated the two maximal contractions. Coefficient of variation for the

measurement of Po averaged 6.2%, with no age, group, or gender difference.

Maximal isokinetic concentric force was determined at 60, 120, 180, 240, 300,

360, 420, 480, and 500°•sec-1. At each velocity the subjects were allowed three warm-

up attempts immediately followed by three maximal concentric contractions, with the

highest force attained for each velocity used for analysis. A two-minute rest period was

given between velocities. Some subjects were not able to produce force at the higher

velocities and/or their measured Vmax was lower than some of the higher measurement

velocities. Coefficient of variation for all concentric velocities, 60 to 500°•sec-1,

averaged 5.3, 3.9, 4.2, 5.5, 5.5, 6.4, 5.8, 7.6, 8.2%, respectively, with no age, group, or

gender difference.

Peak power was calculated for each subject by curve fitting the data of power

(force x velocity) vs. the force produced at each velocity represented as a percentage of

maximal isometric force (%Po). Data to develop the power curve was used if the

measurement velocity was below the measured Vmax for that subject and force

production at two consecutive velocities differed by greater than 4 Nm.

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Statistics

A two-way (age and gender) analysis of variance (ANOVA) was used to compare all

variables. No significant interactions were detected; therefore, post-hoc comparisons

were not completed and the data were collapsed and presented as young and old.

Significance was accepted at p<0.05. Data are presented as mean ± S.E.

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RESULTS

Intramuscular Connective Tissue

Collagen concentration (Young: 9.6±1.1, Old: 10.2±1.2 µg•mg muscle wet wt-1)

and pyridinoline cross-links (Young: 395±65, Old: 351±45 mmol HP•mol collagen-1)

were unchanged (p>0.05) with aging, while AGE’s were increased (p<0.05) by ~200%

(Young: 5.2±1.3, Old: 15.9±4.5 mmol pentosidine•mol collagen-1) with aging (Figure 3).

General and Contractile Proteins

All three general skeletal muscle protein pools were reduced (p<0.05) with aging

(Figure 4). Mixed protein concentration was reduced by 4% (Young: 160±3, Old: 154±1

µg•mg muscle wet wt-1), myofibrillar by 5% (Young: 103±2, Old: 98±2 µg•mg muscle wet

wt-1), and sarcoplasmic by 8% (Young: 64±1, Old: 59±1 µg•mg muscle wet wt-1). The

two main contractile proteins myosin (Young: 46±3, Old: 54±4 µg•mg muscle wet wt-1)

and actin (Young: 21±1, Old: 17±1 µg•mg muscle wet wt-1) were not changed (p>0.05)

with aging. The proportion of MHC I in the muscle increased (P<0.05) with aging, while

MHC IIa and IIx were reduced (p<0.05) (Table 2).

Whole Muscle Characteristics

Quadriceps muscle volume was reduced (p<0.05) by 29% with aging (Table 2).

Quadriceps muscle CSA followed this same trend and was reduced (p<0.05) by 23%

(Young: 62.9±2.9, Old: 48.7±2.3 cm2) (Figure 5). Maximal isometric force (Po) of the

quadriceps was reduced (p<0.05) by 35% (Young: 218±13, Old: 141±11 Nm) and Po

normalized to muscle size was reduced (p<0.05) by 17% (Young: 3.46±0.14, Old:-

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285±0.11 Nm/cm2) (Figure 5). Similarly, peak power of the quadriceps was reduced

(p<0.05) by 48% (Young: 464±36, Old: 243±23 W) and peak power normalized to

muscle CSA was reduced (p<0.05) by 33% (Young: 7.25±0.33, Old: 4.85±0.29 W/cm2)

(Figure 5). Stair climb, chair rise, and walk time were increased by 45 to 80%, and stair

climbing power was reduced by 40% (Table 2) with aging. Quadriceps Vmax was

reduced by 23% (Table 2).

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DISCUSSION

The results of the present study show that endomysial collagen concentration

and enzymatically mediated collagen cross-linking are tightly regulated with aging as

evidenced by the similar concentrations in young and old individuals. However, non-

enzymatically regulated advanced glycation end-product cross-linking is significantly

increased in muscle from healthy sedentary elderly individuals. These results suggest

that the formation of AGE’s over the life span of an individual may contribute to

increased muscle connective tissue protein stiffness and thus contribute to impaired

muscle function in the elderly.

Contrary to our hypotheses, aging had no effect on the intramuscular collagen

concentration. In addition, there was no sex-specific influence on intramuscular

collagen levels. Several recent studies have shown the collagen fraction of skeletal

muscle is much more dynamic that previously thought (3, 32, 33), and the synthesis rate

of intramuscular collagen is elevated in older men (3). Based on these findings and the

collagen concentration data from the current study, collagen breakdown would also

have to be elevated to a similar degree to prevent the accretion of collagen within the

muscle. The discrepancy between the current study and previous animal findings may

be related to the specific portions of the muscle connective tissue studied. Data

obtained from the muscle biopsy technique mainly reflect the endomysial collagen

layers and not the peri- or epimysial layers of connective tissue that have a different

composition of collagen types and roles in the transfer of mechanical forces (20, 23,

37). This is contrary to the animal data which utilizes excised whole muscles for the

analysis of connective tissue. Thus, the potential that biochemical changes are

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occurring in the outer connective tissue layers with aging cannot be excluded. Finally, it

should also be considered that while intramuscular collagen concentration did not

change with aging, it is possible that the type or isoforms of collagen changed. Aging

animal muscle has been shown to alter the collagen isoforms, either by increasing Type

IV collagen (27) or decreasing Type III collagen (16). In these cases, the collagen may

be changing morphology to accommodate a functional need.

To our knowledge, the current investigation is the first to examine the effects of

aging on human skeletal muscle collagen cross-linking. Limited reports exist from post-

mortem analysis of human skeletal muscle that shows the primary mature intramuscular

pyridinium cross-link is HP and lysylpyridinoline (LP) is found only in trace amounts (14,

45). As a result our focus in healthy adult skeletal muscle was limited to that of the

abundant, mature HP species. The existing literature of collagen cross-linking in aging

animals has focused mainly on HP and demonstrates that aging results in a significant

increase in HP with concomitant increases in muscle stiffness (17, 18). The lack of

change seen in aging human muscle may indicate that the normal turnover of skeletal

muscle collagen is robust enough to also turnover mature endomysial connective tissue

cross-links, and these cross-links may not contribute to the reduced muscle function

that occurs with aging.

In contrast to HP collagen cross-linking, the formation of AGE cross-links is not

enzymatically regulated but dictated by the presence of a reducing sugar, the

appropriate protein side chain and oxygen (4). The differences seen in AGE

concentration between young and old in the current investigation are most likely related

to the temporal component of aging. That is, the more time that protein residues have

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23

to come into contact with glucose, there is a greater chance that AGE’s will form. It

should be noted that all the subjects demonstrated normoglycemia and were absent of

overt disease, thus the increased AGE’s noted in the old are reflective of factors other

than altered glucose metabolism, such as that seen in diabetic individuals. The

accumulation of AGE’s in collagenous tissues has been shown to negatively affect

function such as stiffening of the blood vessel walls and kidney structures (4, 35). It is

likely that the 200% increase in pentosidine seen in the older individuals of the current

study influenced tissue stiffness and the passive viscoelastic properties of the muscle

and thus contributed to declines in muscle function. In this context, it should be noted

that pentosidine is commonly used as a surrogate for the many other AGE’s cross-links

(4), and the large increase seen herein is reflective of the accumulation of all possible

AGE’s. The accumulation of AGE’s in the skeletal muscle of the aging women and men

in the current study may be due to the lack of a regular and robust tissue turnover

stimulus such as exercise (19, 31, 33). These concepts warrant further study of AGE

formation in aging human skeletal muscle and the influence of both acute and chronic

exercise.

As the concentrations of mixed, myofibrillar, and sarcoplasmic proteins found in

skeletal muscle are a result of the net turnover (i.e., the sum of protein synthesis and

degradation) (5, 46, 50, 51, 54) and were reduced in the old men and women, there

appears to be a net imbalance between these two processes in aging skeletal muscle.

In addition, the reduction in the myofibrillar protein concentration, in light of the

maintenance of myosin, actin and collagen concentrations (Figure 5), suggests that

other proteins in the myofibrillar apparatus (i.e., titin, nebulin, c-protein, m-protein (37))

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24

are disproportionately lost with aging. Collectively, the current and previous (47) data

collected on the individuals in this study suggest a primary contributor to sarcopenia is

the loss of MHC II fiber number (30) (Table 2). To this end, MHC IIa muscle fibers

produce 5-6 times the normalized power as a MHC I fiber (~9 vs. ~1.5 watts•liter-1) (47).

Thus, an aging individual could afford to lose 5-6 MHC I fibers for every one MHC IIa

fiber lost. Having a higher proportion of MHC I would lend itself to a slower contracting

whole muscle, which is supported by the 23% slower in vivo maximal contraction

velocity in the older subjects. Of course, we cannot rule out the role of a change in the

neural control of muscle with aging (29).

This study and the data reported here are the first to comprehensively examine

the intramuscular connective tissue network in aging men and women. These data

suggest that despite large changes in muscle mass, the concentrations of the two main

contractile proteins, myosin and actin, the protein responsible for the transfer of force

out to the whole muscle, collagen, and the enzymatically regulated cross-linking of

collagen are tightly regulated in aging human skeletal muscle. It does appear that non-

enzymatic addition of advanced glycation endproducts to the intramuscular connective

tissue network may play a role in the reduction of muscle and physical function with

aging.

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25

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ACKNOWLEDGEMENTS

The authors would like to thank the subjects for their participation. This work was

supported by NIH grants R21 AG15833 (TT), K01 AG00831 (TT), M01 RR-14288, and

R01 AG20532 (TT).

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FIGURE LEGENDS

Figure 1. Representative chromatographs for each of the connective tissue assays performed in human skeletal muscle. Elution profiles for collagen and collagen cross-linking were similar between young and old, however aging samples displayed increases in advanced glycation endproducts. HYP: hydroxyproline, PRO: proline, HP: hydroxylysylpyridinoline, PE: pentosidine, IS: internal standard. Figure 2. Standard curve generated from pure collagen using the bicinchoninic acid (BCA) protein assay; y=0.002x + 0.0135. Pure collagen (Sigma C9791) was prepared in 0.1 M acetic acid and absorbance was measured at 540 nm confirming the contribution of collagen to the myofibrillar protein quantification with the BCA assay. Figure 3. Connective tissue characteristics of young and old human skeletal muscle. A) Intramuscular collagen concentration determined from hydroxyproline. B) Intramuscular collagen cross-linking described by the concentration of hydroxylysylpyridinoline (HP). C) Intramuscular advanced glycation endproducts described by the concentration of pentosidine. * p<0.05 from Young. Figure 4. Top: Change in the concentration of the main skeletal muscle pools (mixed, myofibrillar and sarcoplasmic) of the vastus lateralis of young and old individuals. Bottom: Primary components of the assayed myofibrillar fraction (myosin, actin and collagen) are no different between young and old despite decreases in the myofibrillar fraction with aging. *p<0.05 from Young. Figure 5. Comparison between young and old skeletal muscle functional characteristics. CSA: cross-sectional area, Po: Peak isometric force, Po/CSA: Peak isometric force normalized to muscle CSA, Peak Power/CSA: Peak power normalized to muscle CSA. *p<0.05 from Young.

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Table 1. Subject Characterstics.

* P<0.05 from Young. The Young group contained 10 men and 10 women. The Old group contained 10 men and 12 women.

Variable Young (n=20)

Old (n=22)

Age, y 25 ± 1 (22-30) 78 ± 1 (70-93) *

Height, cm 172 ± 2 167 ± 2 *

Weight, kg 70.6 ± 3.2 70.9 ± 2.6

Body fat, % 25 ± 2 32 ± 2 *

Page 32 of 38

Table 2. Functional Ability and Muscle Characterstics.

* P<0.05 from Young.

Variable Young(n=20)

Old(n=22) %∆

Stair ClimbTime, sPower, W

2.57 ± 0.11550 ± 36

4.62 ± 0.33 *334 ± 32 *

+80-40

Chair rise time, s 0.59 ± 0.02 0.92 ± 0.07 * +56

Walk time, s 2.52 ± 0.09 3.65 ± 0.20 * +45

Quadriceps volume, cm3 881 ± 56 622 ± 35 * -29

Quadriceps Vmax, deg/s 660 ± 16 509 ± 11 * -23

Vastus Lateralis Fiber Type% MHC I% MHC IIa% MHC IIx

27 ± 254 ± 219 ± 1

42 ± 3 *45 ± 2 *14 ± 2 *

+55-17-26

Page 33 of 38

Page 34 of 38

R2 = 0.9964

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