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Effect of Low-Magnitude High-Frequency Vibration Treatment on Retardation of Sarcopenia Senescence Accelerated Mice-P8 (SAMP8) Model (doi: 10.1089/rej.2015.1759)

Effect of Low-Magnitude High-Frequency Vibration Treatment on Retardation

of Sarcopenia Senescence Accelerated Mice-P8 (SAMP8) Model

An-yun Guo1, Kwok-sui Leung1,2, Jiang-hui Qin1, Simon Kwoon-ho Chow1,

Wing-hoi Cheung1,2,3*

1
Department of Orthopaedics and Traumatology, Prince of Wales Hospital, The

Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China.
2
Translational Medicine Research & Development Center, Institute of Biomedical and

Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy

of Sciences, China.
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3
The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal

System, The Chinese University of Hong Kong Shenzhen Research Institute,

Shenzhen, PR China.

Word count: 4106

Address for correspondence and reprints:

Wing-Hoi Cheung, PhD, Department of Orthopaedics and Traumatology, 5/F, Clinical

Sciences Building, The Chinese University of Hong Kong, Shatin, New Territories,

Hong Kong SAR, China.

E-mail: louis@ort.cuhk.edu.hk

Telephone: (852)2632-1559; Fax: (852)2632-4618

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ABSTRACT

Background: Sarcopenia related falls and fall-related injuries in community dwelling

elderly garnered more and more interest in recent years. Low-magnitude

high-frequency vibration (LMHFV) was proven beneficial to musculoskeletal system

and recommended for sarcopenia treatment. This study aimed to evaluate the effects

of LMHFV on the sarcopenic animals and explore the mechanism of the stimulatory

effects.

Methods: SAMP8 mice at month 6 were randomized into Ctrl and Vib groups and the

mice in Vib group were given LMHFV (0.3g, 20min/day, 5days/week) treatment. At

month 0, 1, 2, 3, 4 post-treatment, muscle mass, structure and function were assessed.

The potential proliferation capacity of the muscle was also evaluated by investigating
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satellite cells pool and serum myostatin expression.

Results: At late stage, the mice in Vib group showed higher muscle strength (month 4,

p=0.028). Generally, contractibility was significantly improved by LMHFV (CT,

p=0.000; RT50, p=0.000). Enlarged cross-sectional area of fiber type IIA was

observed in Vib group when compared with Ctrl group (p=0.000). No significant

difference of muscle mass was observed. The promotive effect of LMHFV on

myo-regeneration was reflected by suppressed satellite cells pool reduction (month 3,

p=0.000; month 4, p=0.000) and low myostatin expression (p=0.052).

Conclusion: LMHFV significantly improved the structural and functional outcomes

of the skeletal muscle, hence retarding the progress of sarcopenia in SAMP8. It would

be a good recommendation for prevention of the diseases related to skeletal muscle

atrophy.

Key words: LMHFV, SAMP8, Sarcopenia, Skeletal muscle, Satellite cell

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INTRODUCTION

Sarcopenia is an age-related systemic syndrome characterized by the decline of

muscle strength along with the progressive loss of muscle mass, as well as poor

physical performance1, 2. It is well believed that sarcopenia is a normal physiological

process, which is common in elderly over 75 years with high prevalence in both male

(58%) and female (45%)2. All over the world, about 28-35% of elderly are reported to

fall each year3 and sarcopenia is a key risk factor of fall-related injuries among the

community dwelling elderly3-7. Fiatarone's clinical trial with 10 elderly demonstrated

that 8 weeks of high-intensity resistance exercise was effective in increasing skeletal

muscle strength by 74% and muscle size by 9%, hence improving muscle function

and reducing fall incidence8. It is unclear whether different types of exercise yield
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similar results.

Vibration treatment is a non-invasive biophysical modality providing cyclic

loading to whole body transferring mechanical energy to the subjects physically9.

Low-magnitude high-frequency vibration (LMHFV) is a well-accepted intervention

which has been proven to be beneficial to the musculoskeletal system in elderly10, 11. It

has been reported that high frequency vibration treatment increased the skeletal

muscle mass and strength12, 13. Rubin's study demonstrated that LMHFV could inhibit

adipogenesis14. Moreover, our previous studies revealed that LMHFV enhanced bone

fracture healing15-17, angiogenesis and blood flow of the fracture sites18, improved

balancing ability and reduced the fall incidences in community elderly11, 19

. The

expression of myostatin, which is closely related to the satellite cell dysfunction20,

was also reported to be down-regulated by LMHFV21, 22. LMHFV was used in this

study because it meets the safety threshold of ISO-263123 that is safer and more

suitable for the elderly with sarcopenia than high-magnitude therapies (with up to

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50-fold), if applied clinically, while the scientific evidences of high-magnitude ones

should be still relevant.

In this study, we hypothesized that LMHFV stimulated the sarcopenic muscle by

increasing the skeletal muscle mass, improving muscle function, enlarging muscle

satellite cells (SCs) pool and reducing the serum myostatin level in SAMP8 animal

model. The objective of this research was to evaluate the effects of LMHFV on the

sarcopenic animals and explore the related mechanisms.

METHODS

Animal Model and Study Design

Male senescence accelerated mouse P8 (SAMP8) were obtained from the

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Laboratory Animal Service Center (LASEC), the Chinese University of Hong Kong,

where male was used to avoid high hormonal variation24.All animals were kept under

conventional conditions, on a 12:12 hours light:dark cycle with food and water ad

libitum. The research protocol was approved by the Animal Experimentation Ethics

Committee of the Chinese University of Hong Kong (Ref: 12/012/MIS).

A total of 54 SAMP8 mice aged month 6 were randomized into control group

(Ctrl) and vibration group (Vib). The mice in Vib group were treated with LMHFV

(35Hz, 0.3g, where g=gravitational acceleration) 20min/day and 5days/week18, while

those in Ctrl group were given the same regime with the platform power off. The mice

were then euthanized to evaluate the functional and structural outcomes at month 0, 1,

2, 3, 4 post-treatment with 6 mice in each time-point, where Ctrl and Vib groups

shared the month 0 time-point. Along the progression of aging, skeletal muscle fiber

type II was reported to decrease more in sarcopenia25. As a fast-twitch muscle,

gastrocnemius was therefore selected as the target muscle by considering its important

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roles in the posture holding and body movement24.

Body mass of each mouse was measured once a month before euthanasia. At

designated time-points, the mouse was incised under general anaesthesia and

gastrocnemius of the right hindlimb was freshly isolated carefully together with

Achilles's tendon and femur condylar for functional assessment. The assessments,

however, could not be longitudinally performed, which is a limitation. The

contralateral muscle was also isolated for histology and immunofluorescence. After

weighing the wet muscle mass, the isolated left gastrocnemius was put into frozen

2-methylbutane under optimal length for 20seconds and then stored at -80C for the

following histological assessments. In the meantime, blood was drawn from the heart

of the mouse before euthanasia and placed at room temperature for 20 min,
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centrifuged at 3,000rpm for 10 min at 4C. The serum sample was collected and

subsequently stored at -80C for biochemical analysis.

Ex Vivo Functional Test

The isolated left muscle was incubated in the organ bath of the Ringer solution

and the muscle function was investigated with the ex vivo muscle functional test

system (800A, Aurora Scientific Inc, Canada) according to the established protocol24,
26, 27
. After a 15 min stabilization period, two tetanic contractions with 5 min intervals

were induced (1A, 300ms duration, 150Hz stimulation frequency) to activate all the

muscle fibers before the functional test. The optimal length (Lo) of the muscle was

measured by eliciting isometric twitch with increasing muscle length until maximal

force was generated. Under the Lo, the muscle was electronically stimulated two more

times by a single stimulus with 1 min interval to evaluate the twitch characteristic

(twitch force, F0). A continuous stimulus was given three times for 300ms at 150Hz

with 5 min rest to evaluate the tetanic contraction ability (tetanic force, Ft). To

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evaluate the fatigability, pulses of 300ms tetanic stimuli were provided at 150Hz. The

total time of 30 consecutive pulses at 5s intervals was 150s. Skeletal muscle fatigue

was investigated by associating the peak force at a pre-set stimulation time with that

of the initial muscle contraction force induced by a series of electronic stimuli26, 28.

Fatigue induced with repeated tetanic was evaluated by the fatigue rate (FR) and

fatigue extent (percentage loss of muscle force, Loss%)28. The results were recorded

with Dynamic Muscle Control system (DMC v5.4, Aurora Scientific Inc, Canada) and

analyzed by Dynamic Muscle Analysis system (DMA v3.2, Aurora Scientific Inc,

Canada). The muscle cross-sectional area (MCSA) was calculated by dividing the

muscle mass by the muscle optimal length (L0) and the density of mammalian skeletal

muscle (1.06 mg/mm3). Normalized by MCSA, the specific twitch force (SF0) and
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specific tetanic force (SFt) were obtained as the following equations27.

where, D (Muscle density) = 1.06mg/mm3 29

MM = Muscle mass (g): 1000 makes it mg

L0 = Optimal muscle length (cm): 10 makes it mm

Histology

Adenosine triphosphatase (ATPase) staining was performed to identify different

kinds of muscle fibers according to the protocol established in our previous study30.

Muscle transverse sections were cut at 8m with a cryostat (Cryocut 1800, Leica,

Germany) at -20C. All sections were brought to room temperature and incubated in

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pre-incubating solution at pH4.45 for exactly 5 min and then incubated in ATP

solution at pH9.4 for 25 min. Three random fields of each section were taken from

each section with a microscope (Leica Microsystems Ltd) at 50 and

200magnifications. Morphometric analysis was performed with Image-Pro Plus

Software (Version 6.0, Media Cybernetics, Inc. USA). The muscle fiber could be

identified as type I (darkest), type IIA (lightest) and type IIB (moderate). The average

fiber size was quantified for each fiber type.

Immunofluorescence

Pax7/laminin double staining was performed on the muscle cryo-sections to

identify the SCs. The Pax7 antibodies (primary: rabbit anti-mouse Pax7, 1:10, Abcam,

UK; secondary: goat-anti rabbit GaM-Biotin, 1:1000, JIRL, USA; tertiary: Cy3-strep,
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1:2500, JIRL, USA) and laminin antibodies (primary: rabbit anti-mouse, 1:800,

Abcam, UK; secondary: goat anti-rabbit, IgG H&L, 1:500, Abcam, UK) were used

based on Diao et al. protocol31. Fluorescence microscope system (DM 6000B upright

microscope, Leica-micro system, GmbH, Werzlar, Germany) and IM50 software

(Leica-micro system, GmbH, Werzlar, Germany) were used for spatial detection of the

SCs and analysis. Each muscle section was divided into 4 parts and one field was

captured randomly in each part. The SC was defined as the Pax7+/DAPI+ cell (pink

fluorescent signal) located between the laminin basement membrane (green

fluorescent signal) and the muscle fiber. The SC number and the muscle fiber number

were counted by Image-Pro Plus software (Media Cybernetics, Inc.) and the number

of SC per 100 muscle fibers was regarded as the parameter for SC pool evaluation32.

Enzyme-Linked Immunosorbent Assay(ELISA)

The myostatin level in serum was determined by mouse myostatin ELISA kit

according to manufacturers instructions. The absorbance was read at 450nm with

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microplate spectrophotometer (Quant, BioTek Instruments, Inc. Vermont, USA) and

Gen5 software (Gen5 Version1.08, BioTek Instruments, Inc. Vermont, USA) was used

to calculate the density of myostatin in the serum. However, myostatin level was not

corrected by the change of blood volume, as the systemic effect of LMHFV on

systemic blood volume was assumed not significant.

Statistical Analysis

All the quantitative data were expressed in mean standard deviation (SD). Data

analysis was performed with two-way analysis of variance (ANOVA) and post-hoc

Tukey tests using SPSS (Version 20.0, IBM, USA) for between-group comparison

among different time-points, where one-way ANOVA and post-hoc Tukey test were

used for within-group comparison. Independent-sample t test was performed to

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compare the parameters between the two groups at each time-point. The linear slope

was calculated for fatigue rate (FR) analysis and one-way ANOVA followed by Tukey

post-hoc test were performed for the slope evaluation. The significance level was set

at p0.05.

RESULTS

Muscle Mass and Cross-Sectional Area

The largest muscle mass (MM) appeared at month 1 (Ctrl: 0.1370.007g; Vib:

0.1320.008g) and decreased from month 1 to month 4 (Figure 1A). In Vib group,

MM at month 4 was lower than those of month 1 and month 3 significantly (p=0.024

and p=0.043 respectively, one-way ANOVA). There was no significant difference

between Ctrl and Vib groups in MM (p=0.285, two-way ANOVA). The change of

muscle cross-sectional area (MCSA) in Ctrl group was not significant (Figure 1B).

However, in Vib group, the MCSA at month 4 was much lower than that at month 1

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and month 2 significantly (p=0.029 and p=0.001 respectively, one-way ANOVA). No

significant difference was observed between Ctrl and Vib groups (p=0.218, two-way

ANOVA).

Muscle Strength

Muscle strength was measured at month 0, 1, 2, 3 and 4 after vibration treatment

and normalized by the MCSA to derive the specific twitch force (SF0) and specific

tetanic force (SFt). In Ctrl group, the SF0 increased from month 0 to month 3 (p=0.175,

one-way ANOVA) but then decreased slightly from month 3 to month 4 (Figure 2A).

In Vib group, the peak of SF0 appeared at month 4. From month 0 to month 4, the SF0

showed an increasing trend (p=0.166, one-way ANOVA). The SF0 in Ctrl group was

higher than that in the Vib group from month 1 to month 3, but became significantly
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lower at month 4 (Ctrl <Vib, p=0.028, independent-sample t test). The peak of SFt in

Ctrl group was observed at month 1 (Figure 2B). From month 1 to month 3, there was

no significant change but a decrease at month 4. Compared with the month 0, SFt of

Ctrl group at month 1, 2 and 3 were significantly higher (p=0.001, 0.004 and 0.003

respectively, one-way ANOVA). The SFt in Vib group showed an increasing trend

from month 0 to month 4 (p=0.198, one-way ANOVA). From month 1 to month 3, the

SFt in Ctrl group were higher than those in Vib group, with significant difference at

month 1 (p=0.008, independent-sample t test). But the relative relationship was

reversed at month 4. SFt in Ctrl group was slightly lower than that in Vib group

(p=0.315, independent-sample t test).

Contractibility and Fatigability

There was no significant difference among different time-points within group in

both Ctrl and Vib groups. In general, the CT in Ctrl group was longer than that in Vib

group (p=0.000, two-way ANOVA) and significant differences were observed at

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month 2 (Ctrl>Vib, p=0.000, independent-sample t test) and month 4 (Ctrl>Vib,

p=0.005, independent-sample t test) (Figure 3A). Ctrl group showed longer RT50 than

Vib group generally (p=0.000, two-way ANOVA). Moreover, significant differences

were observed at month 1 (Ctrl>Vib, p=0.002, independent-sample t test) and month 2

(Ctrl>Vib, p=0.000, independent-sample t test) (Figure 3B).

In both Ctrl and Vib groups, no obvious difference of FR or Loss% was observed

among different time-points within group. Generally, FR in Ctrl groups was higher

than that in the Vib group (p=0.01, two-way ANOVA). At month 1, the difference was

significant (p=0.001, independent-sample t test) (Figure 3C). In Vib group, no

significant change of Loss% among different time-points was observed in Ctrl group.

The Loss% in Vib group was lower than that in the Ctrl group (p=0.01, two-way
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ANOVA) with significant difference at month 1 (p=0.035, independent-sample t test)

(Figure 3D).

Fiber Typing and Fiber Cross Sectional Area (FCSA)

In Ctrl group, the largest FCSA of fiber type I appeared at month 2, which was

higher than those at month 3 and month 4 (p=0.009 and p=0.009 respectively,

one-way ANOVA) (Figure 4A). In Vib group, the FCSA at month 1 and month 4 was

significantly larger than those at the corresponding time-points in Ctrl group (p=0.008

and p=0.042 respectively, independent-sample t test), but a lower FCSA at month 2

when compared with the Ctrl group (p=0.001, independent-sample t test). The peak of

FCSA type IIA in Ctrl group was at month 3, with no significant difference among the

time-points within group (Figure 4B). In Vib group, FCSA increased from month 0 to

month 1 (p=0.000, one-way ANOVA), then followed with decrease from month 1 to

month 4 (month 1 > month 2, p=0.017; month 1 > month 4, p=0.000, one-way

ANOVA). In general, Vib group had larger FCSA of type IIA than Ctrl group

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(p=0.000, two-way ANOVA) and the differences were significant at month 1, 2 and 3

(p=0.000, p=0.035 and p=0.003 respectively, independent-sample t test). There was

no significant within-group change of FCSA of type IIB among different time-points

in both Ctrl and Vib groups; no significant difference was observed between Ctrl and

Vib groups at each time-point (Figure 4C).

SC Counting

From month 2 to month 4, the SC amount sharply decreased in Ctrl group, and

the SC counting at month 3 and month 4 were significantly lower than month 2

(p=0.000 and 0.000 respectively, one-way ANOVA) (Figure 5B). Vib group had

higher SC amount at month 3 and month 4 than those at the corresponding time-points

in Ctrl group (p=0.001 and p=0.001 respectively, independent-sample t test).

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Generally, the SC amount in Vib groups was higher than Ctrl group (p=0.001,

two-way ANOVA).

Serum Myostatin Level

Myostatin level decreased from month 0 to month 2 and increased from month 2

to month 4 in both Ctrl and Vib groups, but the difference was not significant among

the time-points within group (Figure 6). In general, Vib group had higher serum

myostatin level than Ctrl and the difference was significant at month 2 (p=0.031,

independent-sample t test). However, a significantly lower expression of myostatin in

Vib group was observed at month 4 when compared with Ctrl group (p=0.050,

independent-sample t test).

DISCUSSION

The present study aims to investigate the effects of LMHFV on progression of

sarcopenia in the SAMP8 mice. The current results showed that muscle fiber type IIA,

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which accounted for more than 50% of the gastrocnemius, was significantly improved

by LMHFV in FCSA. LMHFV could also enhance muscle function, such as

contractility and fatigue durability. Compared with Ctrl group, the increased satellite

cell pool and lower myostatin level at late time-point in Vib group indicated that

LMHFV could suppress the age-related decrease of skeletal muscle regeneration.

The long-term effect of LMHFV on skeletal muscle function was proven positive.

It was reported that 10 weeks of high frequency vibration (20-50Hz, 2-4mm)

treatment increased the skeletal muscle strength12, 13. Previous study revealed that 10

min of whole body vibration (WBV) (45Hz, 2.2mm, 7.7g) induced lower running

capacity after 2 min post-treatment33. However, some other investigations found that

over 5 months of high frequency (>30Hz, <2-4mm) WBV treatment significantly

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improved muscle strength and power34, 35. In the present study, LMHFV treatment

caused lower SF0 and SFt in Vib group from month 0 to month 3. However, the SF0 in

Vib group was higher than that in the Ctrl group at month 4, and a similar trend of SFt

was also observed. Our results also demonstrated that the SF0 and SFt in Vib group

showed increasing trend from month 0 to month 4. These proved that the acute effect

of vibration treatment on skeletal strength was negative but the long-term effect was

positive.

In this study, LMHFV improved the muscle contractility by shortening CT and

RT50 at each time-point in Vib group. It has been demonstrated by previous studies

that vibration (35-40Hz) was beneficial in improving the skeletal muscle contractility

with regard to the reduction of response time and improvement of balancing

ability36-38. We know that skeletal muscle fiber type IIA plays a crucial role in

movement and balance with fast twitch speed and high fatigue durability39. One of our

previous studies demonstrated that high-frequency vibration treatment (20Hz,

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20min/day, 5days/week) significantly improved the gait speed19. Also, the balancing

ability was improved and fall incidence was reduced after 18 months of LMHFV

treatment (35Hz, 0.3g, 20min/day, 5days/week) in our randomized controlled trial11.

These evidences support that LMHFV would be beneficial to skeletal muscle fiber

type IIA with a positive long-term effect on the community elderly. The fiber type IIA

was more sensitive to the mechanical stimulation and vertical vibration treatment was

reported to improve the contractibility of gastrocnemius by provoking the contractile

element40. The improved muscle contractility provided a good evidence to support the

beneficial effect of LMHFV on enhancing the muscular performance by shortening

response time and improving balancing ability. Moreover, it shows a clinical potential

in the application of LMHFV on muscle function decline in the community elderly.

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Both of the FR and Loss% were significantly reduced by LMHFV in this study,

which indicated that LMHFV enhanced the fatigue resistance of skeletal muscle. It

has been proven that vibration (25Hz, 4mm) enhanced the skeletal muscle endurance

by improving the endurance-associated parameters such as energy metabolism and

turnover, blood flow and neuromuscular activation37. Hilgers et al. also reported that

vibration training (30Hz, 1-2mm) improved the walking endurance in sit-to-stand test,

timed up and go test, 10-meter walk test and 6-minute walk test41. This is well known

that muscle fiber type IIA is fast-twitch and fatigue resistance, hence the improved

fatigue durability induced by LMHFV may be due to the increased muscle fiber type

IIA as shown in our muscle fiber typing results.

Previous study revealed that consistent mechanical loading induced an increased

muscle fiber type IIA MHC expression42. Moreover, WBV (50Hz, 2.5mm) was also

proven to improve the fiber type IIA motor units (fast fatigue resistant motor units, FR

MUs)43. In the present study, it was revealed that the stimulatory effect of LMHFV on

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the FCSA of fiber type IIA was present throughout the whole treatment process.

However, the improvement of LMHFV on muscle strength occurred mainly at the late

phase. The explanation for the asynchronized effects of LMHFV on muscle fiber size

and muscle strength might be due to potential loading injury in weakened muscle at

initial treatment period that was caused by myofiber disruption due to stretching or

increased muscle strain when they are loaded and lengthened.44. The target muscle in

present study was gastrocnemius, which was mainly composed of fiber type IIA as

contractile tissue24. We found that the FCSA of fiber type IIA was significantly

improved by LMHFV. It was also reported that vibration treatment (45Hz, 0.3g) could

increase muscle fiber type II FCSA by 29%45. Though no significant change of

muscle mass was observed, LMHFV showed beneficial effect on skeletal muscle in
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consideration of the improved function and type IIA FCSA. The relationship between

muscle mass and muscle quality was not linear and muscle mass might not reflect the

real muscle quality well46. Von Stengel's study indicated that vibration (25-35Hz,

1.7-2.0mm) treatment could alter the body composition by down-regulating the

distribution of fat in trunk and lower limbs47. Moreover, Rubin's study demonstrated

that LMHFV (90Hz, 12m, 0.2g) could inhibit adipogenesis14. We therefore

postulated that LMHFV improved the muscle quality by altering muscle composition

in increasing contractile but not enlarging the muscle mass directly.

SCs are regarded as stem cell-like cells which are responsible for skeletal muscle

repair and regeneration48. Besides, SCs were reported to be responsible for skeletal

muscle hypertrophic growth48. The maintenance of quiescent SCs was a requisite for

structural integrity of muscle fibers48. Normally, reduction of SCs was accompanied

with skeletal muscle atrophy49. It has been documented that mechanical stimulation or

resistant exercise could increase the number of SCs48, 50 and newly formed SCs would

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incorporate and fuse into the muscle fibers48. The incorporation of new skeletal

myonuclei from SCs proceeded the subsequent muscle fiber growth51. Therefore, the

presence of SCs was essential to maintain the skeletal muscle mass. Our results

showed that the decreasing trend of SCs in Ctrl group was reduced at late stage.

Rubin's study on C57BL/6 mice also confirmed that low intensity vibration treatment

(90Hz, 0.3g) could significantly retard ovariectomy-induced satellite cell reduction22.

Therefore, the delayed expansion of SCs pool after vibration treatment might indicate

a delayed promotion effect of LMHFV on skeletal muscle self-repair and regeneration.

Myostatin was also proven to associate with muscle stem cell dysfunction and a

higher myostatin expression indicates an impaired myogenic capacity20. As a negative

regulator, myostatin was reported to affect skeletal muscle growth and regulate the
Rejuvenation Research

SCs activation48, 52. Based on our results and the above evidence21, LMHFV-induced

early higher but late lower myostatin level indicated the early reduction but late

promotion effect of LMHFV on skeletal muscle self-repair capacity.

There were a few limitations in this study. Firstly, Takeda's research reported that

the average lifespan of SAMP8 was 9.7 months53 and our previous study found that

the onset time of sarcopenia in SAMP8 was month 824. Therefore, the last time-point

of this study was selected at month 4 post-treatment when the animals were 10

months old. However, we found that the muscle strength of SAMP8 in Vib group

showed a consistent increasing trend. Recruiting one more time-point at month 5

post-treatment might present a clearer picture. Secondly, serum myostatin expression

could reflect the systemic myostatin level to some extent but local skeletal muscle

myostatin expression would be more accurate to reveal the relationship between

skeletal muscle function and myostatin expression. In the future,

immunohistochemistry and PCR of local skeletal muscle myostatin investigation

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Effect of Low-Magnitude High-Frequency Vibration Treatment on Retardation of Sarcopenia Senescence Accelerated Mice-P8 (SAMP8) Model (doi: 10.1089/rej.2015.1759)

would be performed instead of serum myostatin assessment with ELISA.

In conclusion, the effect of LMHFV on skeletal muscle function was positive.

The reduction of twitch force and tetanic force at early stage and the increase at late

stage indicated that the acute effect of LMHFV on muscle force was negative but the

long-term effect was positive. Furthermore, the vibration treatment improved the

skeletal muscle contractibility and endurance, and maintained SCs pool. The muscle

fiber type IIA was more sensitive to LMHFV compared with other types of muscle

fibers. Although no visible improvement of muscle mass was observed, the fiber

distribution of the gastrocnemius was changed by the vibration treatment, which

indicated that LMHFV improved the skeletal muscle function by optimizing the

muscle composition but not increasing the muscle mass directly. Based on the
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diagnostic criteria proposed by the European Working Group on Sarcopenia in Older

People (EWGSOP) in 2010, we are unable to demonstrate that LMHFV reverses

sarcopenia progression. Nonetheless, LMHFV could retard the decrease of skeletal

muscle function with no adverse effects. Most importantly, LMHFV (0.3g, 35Hz)

meets the safety threshold of ISO-263123 and our results confirm that LMHFV

induces comparable responses to that of high-magnitude exercise, which is very

suitable for elderly population54, 55. This non-invasive physical intervention would be

a good recommendation for prevention program in falls and fall-related injuries in

community dwelling elderly.

ACKNOWLEDGEMENTS

This work was supported by General Research Fund (Ref: 14103314 and 469911),

Research Grants Council, HKSAR. The author would like to show gratitude to Dr.

Wang Lijun for helping in the immunofluorescence with Pax7/laminin on satellite

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cell.

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REFERENCES

1. Cruz-Jentoft AJ, Baeyens JP, Bauer JM, et al. Sarcopenia: European consensus
on definition and diagnosis: Report of the European Working Group on
Sarcopenia in Older People. Age Ageing. Jul 2010;39(4):412-423.
2. Kim TN, Choi KM. Sarcopenia: definition, epidemiology, and
pathophysiology. J Bone Metab. May 2013;20(1):1-10.
3. Woo N, Kim SH. Sarcopenia influences fall-related injuries in
community-dwelling older adults. Geriatr Nurs. Jul-Aug 2014;35(4):279-282.
4. Rom O, Kaisari S, Aizenbud D, Reznick AZ. Lifestyle and sarcopenia-etiology,
prevention, and treatment. Rambam Maimonides Med J. Oct 2012;3(4):e0024.
5. Muscaritoli M, Anker SD, Argiles J, et al. Consensus definition of sarcopenia,
cachexia and pre-cachexia: joint document elaborated by Special Interest
Groups (SIG) "cachexia-anorexia in chronic wasting diseases" and "nutrition
in geriatrics". Clin Nutr. Apr 2010;29(2):154-159.
6. Morley JE, von Haehling S, Anker SD, Vellas B. From sarcopenia to frailty: a
road less traveled. J Cachexia Sarcopenia Muscle. Mar 2014;5(1):5-8.
7. Tanimoto Y, Watanabe M, Sun W, et al. Sarcopenia and falls in
community-dwelling elderly subjects in Japan: Defining sarcopenia according
to criteria of the European Working Group on Sarcopenia in Older People.
Arch Gerontol Geriatr. Sep-Oct 2014;59(2):295-299.
8. Fiatarone MA, Marks EC, Ryan ND, et al. High-Intensity Strength Training in
Rejuvenation Research

Nonagenarians. Effects on skelatal muscle. JAMA. 1990;263(22):3029-3024.

9. Rittweger J. Vibration as an exercise modality: how it may work, and what its
potential might be. Eur J Appl Physiol. Mar 2010;108(5):877-904.
10. Judex S, Rubin CT. Is bone formation induced by high-frequency mechanical
signals modulated by muscle activity. J Musculoskelet Neuronal Interact.
2010;10(1):3-11.
11. Leung KS, Li CY, Tse YK, et al. Effects of 18-month low-magnitude
high-frequency vibration on fall rate and fracture risks in 710 community
elderly--a cluster-randomized controlled trial. Osteoporos Int. Jun
2014;25(6):1785-1795.
12. Machado A, Garcia-Lopez D, Gonzalez-Gallego J, Garatachea N. Whole-body
vibration training increases muscle strength and mass in older women: a
randomized-controlled trial. Scand J Med Sci Sports. Apr 2010;20(2):200-207.
13. Colson SS, Petit PD. Lower limbs power and stiffness after whole-body
vibration. Int J Sports Med. Apr 2013;34(4):318-323.
14. Rubin CT, Capilla E, Luu YK, et al. Adipogenesis is inhibited by brief, daily
exposure to high-frequency, extremely low-magnitude mechanical signals.
Proc Natl Acad Sci U S A. Nov 6 2007;104(45):17879-17884.
15. Shi HF, Cheung WH, Qin L, et al. Low-magnitude high-frequency vibration
treatment augments fracture healing in ovariectomy-induced osteoporotic bone.
Bone. May 2010;46(5):1299-1305.
16. Chow DH, Leung KS, Qin L, et al. Low-magnitude high-frequency vibration
(LMHFV) enhances bone remodeling in osteoporotic rat femoral fracture
healing. J Orthop Res. May 2011;29(5):746-752.
17. Leung KS, Shi HF, Cheung WH, et al. Low-magnitude high-frequency
vibration accelerates callus formation, mineralization, and fracture healing in
rats. J Orthop Res. Apr 2009;27(4):458-465.
18. Cheung WH, Sun MH, Zheng YP, et al. Stimulated angiogenesis for fracture

18
 Page 19 of 28

19
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Effect of Low-Magnitude High-Frequency Vibration Treatment on Retardation of Sarcopenia Senescence Accelerated Mice-P8 (SAMP8) Model (doi: 10.1089/rej.2015.1759)

healing augmented by low-magnitude, high-frequency vibration in a rat

model-evaluation of pulsed-wave doppler, 3-D power Doppler
ultrasonography and micro-CT microangiography. Ultrasound Med Biol. Dec
2012;38(12):2120-2129.
19. Cheung WH, Mok HW, Qin L, et al. High-frequency whole-body vibration
improves balancing ability in elderly women. Arch Phys Med Rehabil. Jul
2007;88(7):852-857.
20. McKay BR, Ogborn DI, Bellamy LM, et al. Myostatin is associated with
age-related human muscle stem cell dysfunction. FASEB J. Jun
2012;26(6):2509-2521.
21. Ceccarelli G, Benedetti L, Galli D, et al. Low-amplitude high frequency
vibration down-regulates myostatin and atrogin-1 expression, two components
of the atrophy pathway in muscle cells. J Tissue Eng Regen Med. May
2014;8(5):396-406.
22. Ferechette DM, Krishnamoorthy D, Adler BJ, et al. Diminished satellite cells
and elevated adipogenic gene expression in muscle and caused by ovariectomy
are averted by low-magnitude mechanical signals. J Appl Physiol.
2015;199:27-36.
23. Muir J, Kiel DP, Rubin CT. Safety and severity of accelerations delivered from
whole body vibration exercise devices to standing adults. J Sci Med Sport.
Nov 2013;16(6):526-531.
24. Guo AY, Leung KS, Siu MF, et al. Muscle Mass, Structural and Functional
Rejuvenation Research

Investigations of Senescence-accelerated Mouse P8 (SAMP8). Exp. Anim.

2015:(in press).
25. Lee WS, Cheung WH, Qin L, et al. Age-associated decrease of type IIA/B
human skeletal muscle fibers. Clin Orthop Relat Res. Sep 2006;450:231-237.
26. Derave W, Eijnde BO, Ramaekers M, Hespel P. Soleus muscles of SAMP8
mice provide an accelerated model of skeletal muscle senescence. Exp
Gerontol. Jul 2005;40(7):562-572.
27. Moorwood C, Liu M, Tian Z, Barton ER. Isometric and eccentric force
generation assessment of skeletal muscles isolated from murine models of
muscular dystrophies. J Vis Exp. 2013(71):e50036.
28. Cairns SP, Robinson DM, Loiselle DS. Double-sigmoid model for fitting
fatigue profiles in mouse fast- and slow-twitch muscle. Exp Physiol. Jul
2008;93(7):851-862.
29. Payne AM, Dodd SL, Leeuwenburgh C. Life-long calorie restriction in Fischer
344 rats attenuates age-related loss in skeletal muscle-specific force and
reduces extracellular space. Journal of applied physiology (Bethesda, Md. :
1985). Dec 2003;95(6):2554-2562.
30. Cheung WH, Lee WS, Qin L, et al. Type IIB human skeletal muscle fibers
positively correlate with bone mineral density irrespective to age. Chin Med J
(Engl). Nov 2010;123(21):3009-3014.
31. Diao Y, Guo X, Li Y, et al. Pax3/7BP is a Pax7- and Pax3-binding protein that
regulates the proliferation of muscle precursor cells by an epigenetic
mechanism. Cell Stem Cell. Aug 3 2012;11(2):231-241.
32. Mackey AL, Kjaer M, Charifi N, et al. Assessment of satellite cell number and
activity status in human skeletal muscle biopsies. Muscle Nerve. Sep
2009;40(3):455-465.
33. Padulo J, Filingeri D, Chamari K, et al. Acute effects of whole-body vibration
on running gait in marathon runners. J Sports Sci. 2014;32(12):1120-1126.

19
 Page 20 of 28

20
This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Effect of Low-Magnitude High-Frequency Vibration Treatment on Retardation of Sarcopenia Senescence Accelerated Mice-P8 (SAMP8) Model (doi: 10.1089/rej.2015.1759)

34. Wheeler AA, Jacobson BH. Effect of whole-body vibration on delayed onset
muscular soreness, flexibility, and power. J Strength Cond Res.
2013;27(9):2527-2532.
35. Osawa Y, Oguma Y, Ishii N. The effects of whole-body vibration on muscle
strength and power: a meta-analysis. J Musculoskelet Neuronal Interact.
2013;13(3):380-390.
36. Lam FM, Lau RW, Chung RC, Pang MY. The effect of whole body vibration
on balance, mobility and falls in older adults: a systematic review and
meta-analysis. Maturitas. Jul 2012;72(3):206-213.
37. Ritzmann R, Kramer A, Bernhardt S, Gollhofer A. Whole body vibration
training--improving balance control and muscle endurance. PLoS One.
2014;9(2):e89905.
38. Despina T, George D, George T, et al. Short-term effect of whole-body
vibration training on balance, flexibility and lower limb explosive strength in
elite rhythmic gymnasts. Hum Mov Sci. Feb 2014;33:149-158.
39. Schiaffino S, Reggiani C. Fiber types in mammalian skeletal muscles. Physiol
Rev. 2011;91:1447-1531.
40. ochyski D, Kaczmarek D, Rdowicz MJ, et al. Long-term effects of
whole-body vibration on motor unit contractile function and myosin heavy
chain composition in the rat medial gastrocnemius. J Musculoskelet Neuronal
Interact. 2013;13(4):430-441.
41. Hilgers C, Mundermann A, Riehle H, Dettmers C. Effects of whole-body
Rejuvenation Research

vibration training on physical function in patients with multiple sclerosis.

NeuroRehabilitation. 2013;32(3):655-663.
42. Kenneth MB, Fadia H. Effects of different activity and inactivity paradigms on
myosin heavy chain gene expression in striated muscle. J Appl Physiol.
2001;90(1):345-357.
43. Lochynski D, Baczyk M, Kaczmarek D, et al. Adaptations in physiological
properties of rat motor units following 5 weeks of whole-body vibration. Appl
Physiol Nutr Metab. Sep 2013;38(9):913-921.
44. Sun KT, Leung KS, Siu PM, et al. Differential effects of low-magnitude
high-frequency vibration on reloading hind-limb soleus and gastrocnemius
medialis muscles in 28-day tail-suspended rats. J Musculoskelet Neuronal
Interact. 2015:(in press).
45. Xie L, Rubin C, Judex S. Enhancement of the adolescent murine
musculoskeletal system using low-level mechanical vibrations. J Appl Physiol.
2008;104(4):1056-1062.
46. Karsten K, Martin E. Strength and muscle mass loss with aging process. Age
and strength loss. Muscles Ligaments Tendons J. 2014;3(4):346-350.
47. von Stengel S, Kemmler W, Engelke K, Kalender WA. Effect of whole-body
vibration on neuromuscular performance and body composition for females 65
years and older: a randomized-controlled trial. Scand J Med Sci Sports. Feb
2012;22(1):119-127.
48. Blaauw B, Reggiani C. The role of satellite cells in muscle hypertrophy. J
Muscle Res Cell Motil. Feb 2014;35(1):3-10.
49. Buckingham M, Montarras D. Skeletal muscle stem cells. Curr Opin Genet
Dev. Aug 2008;18(4):330-336.
50. Macaluso F, Myburgh KH. Current evidence that exercise can increase the
number of adult stem cells. J Muscle Res Cell Motil. Aug
2012;33(3-4):187-198.

20
 Page 21 of 28

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This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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51. Bruusgaard JC, Johansen IB, Egner IM, et al. Myonuclei acquired by overload
exercise precede hypertrophy and are not lost on detraining. Proc Natl Acad
Sci U S A. Aug 24 2010;107(34):15111-15116.
52. Wang M, Yu H, Kim YS, et al. Myostatin facilitates slow and inhibits fast
myosin heavy chain expression during myogenic differentiation. Biochem
Biophys Res Commun. Sep 14 2012;426(1):83-88.
53. Takeda T, Hosokawa M, Higuchi K. Senescence-accelerated mouse (SAM): a
novel murine model of senescence. Exp Gerontol. 1997;32:105-109.
54. Chan ME, Uzer G, Rubin CT. The potential benefits and inherent risks of
vibration as a non-drug therapy for the prevention and treatment of
osteoporosis. Current osteoporosis reports. Mar 2013;11(1):36-44.
55. Thompson WR, Yen SS, Rubin J. Vibration therapy: clinical applications in
bone. Current opinion in endocrinology, diabetes, and obesity. Dec
2014;21(6):447-453.
Rejuvenation Research

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Figure Legends
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Effect of Low-Magnitude High-Frequency Vibration Treatment on Retardation of Sarcopenia Senescence Accelerated Mice-P8 (SAMP8) Model (doi: 10.1089/rej.2015.1759)

Figure 1. Morphological parameters between Ctrl and Vib groups. A: The peak MM

in Ctrl and Vib groups were observed at month 1 (0.1370.007g, 0.1320.008g

respectively), and decreased from month 1 to month 4. No significant difference

between Ctrl and Vib groups was found (p=0.285, two-way ANOVA). B: In Vib group,

the MCSA at month 4 was much lower than those in month 1 and month 2
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significantly (p=0.029 and p=0.001 respectively, one-way ANOVA). No significant

difference was observed between Ctrl and Vib groups (p=0.218, two-way ANOVA).

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Effect of Low-Magnitude High-Frequency Vibration Treatment on Retardation of Sarcopenia Senescence Accelerated Mice-P8 (SAMP8) Model (doi: 10.1089/rej.2015.1759)

Figure 2. Muscle strength. A: From month 0 to month 4, SF0 showed an increasing

trend in Vib group. The SF0 in Ctrl group was higher than those in the Vib group from

month 1 to month 3, but became significantly lower at month 4 (Ctrl <Vib, p=0.028,

independent-sample t test). B: From month 1 to month 3, SFt in Ctrl group was higher

than those in Vib group, with significant difference at month 1 (p=0.008,

Rejuvenation Research

independent-sample t test). But the relative relationship was reversed at month 4.

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Effect of Low-Magnitude High-Frequency Vibration Treatment on Retardation of Sarcopenia Senescence Accelerated Mice-P8 (SAMP8) Model (doi: 10.1089/rej.2015.1759)
Rejuvenation Research

Figure 3. Muscle contractibility and fatigability. A: CT of Vib group was reduced by

LMHFV when compared with Ctrl group (p=0.000, two-way ANOVA), with

significant differences at month 2 and month 4; B: RT50 in Vib group was lower than

Ctrl group (p=0.000, two-way ANOVA), with significant difference at month 1 and

month 2; C: In general, FR in Vib group was lower than Ctrl group (p=0.010,

two-way ANOVA) with significant difference at month 1; D: The loss% in Vib group

was lower than Ctrl group (p=0.010, two-way ANOVA) generally and the difference

was significant at month 1.

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Rejuvenation Research

Figure 4. Fiber cross-sectional area of the muscle. A: Muscle fiber type I: the FCSA

in Vib group was larger than Ctrl group at the late phase; B: Muscle fiber type IIA:

the peak of FCSA in Ctrl group was at month 3 and that in Vib group was at month 1.

The FCSA in Vib group was larger than Ctrl group generally (p=0.000, two-way

ANOVA). C: Muscle fiber type IIB: No significant difference was observed within or

between groups.

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Effect of Low-Magnitude High-Frequency Vibration Treatment on Retardation of Sarcopenia Senescence Accelerated Mice-P8 (SAMP8) Model (doi: 10.1089/rej.2015.1759)
Rejuvenation Research

Figure 5. Satellite cells counting. A: Immunofluorescence with Pax7/laminin (200)

shows the SCs amount of the muscle in Ctrl and Vib groups at different time-points. B:

Generally, the decrease of SCs amount was significantly suppressed by LMHFV

treatment (p=0.001, two-way ANOVA). In Ctrl group, the SCs amount decreased from

month 3 with significant difference at month 3 and month 4 (p=0.000 and p=0.000

respectively).

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Effect of Low-Magnitude High-Frequency Vibration Treatment on Retardation of Sarcopenia Senescence Accelerated Mice-P8 (SAMP8) Model (doi: 10.1089/rej.2015.1759)
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independent-sample test) but higher at month 4 (p=0.052, independent-sample t test).
myostatin level in Ctrl group was lower than Vib group at month 2 (p=0.031,
28

Figure 6. Serum myostatin level in Ctrl and Vib groups at different time-points. The
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