C-terminal Agrin Fragment as a potential marker for sarcopenia caused by degeneration of the neuromuscular junction

Experimental Gerontology 48 (2013) 76–80

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C-terminal Agrin Fragment as a potential marker for sarcopenia caused by degeneration of the neuromuscular junction M. Drey a,⁎, C.C. Sieber a, J.M. Bauer a, b, W. Uter c, P. Dahinden d, R.G. Fariello d, J.W. Vrijbloed d and the FiAT intervention group 1 a

Universität Erlangen-Nürnberg, Institute for Biomedicine of Aging, Heimerichstrasse 58, 90419 Nuremberg, Germany Geriatric Centre Oldenburg, Rahel-Straus-Str. 10, 26133 Oldenburg, Germany Universität Erlangen-Nürnberg, Department of Medical Informatics, Biometry and Epidemiology, Waldstrasse 6, 91054 Erlangen, Germany d Neurotune AG, Wagistrasse 27a, 8952 Schlieren, Switzerland b c

a r t i c l e

i n f o

Article history: Received 26 December 2011 Received in revised form 27 April 2012 Accepted 30 May 2012 Available online 7 June 2012 Section Editor: A. Simm Keywords: Sarcopenia Neuromuscular junction Agrin Neurotrypsin Exercise Vitamin D

a b s t r a c t Introduction: Sarcopenia is considered to be an enormous burden for both the individuals affected and for society at large. A multifactorial aetiology of this geriatric syndrome has been discussed. Amongst other pathomechanisms, the degeneration of the neuromuscular junction (NMJ) may be of major relevance. The intact balance between the pro-synaptic agent agrin and the anti-synaptic agent neurotrypsin ensures a structurally and functionally intact NMJ. Excessive cleavage of the native motoneuron-derived agrin by neurotrypsin into a C-terminal Agrin Fragment (CAF) leads to functional disintegration at the NMJ and may consecutively cause sarcopenia. The present study evaluates the hypothesis that CAF serum concentration is a potential marker for the loss of appendicular lean mass in older adults. It also explores how CAF concentration is influenced by vitamin D supplementation and physical exercise. Method: Serum was taken from 69 (47 female) prefrail community-dwelling older adults participating in a training intervention study to measure the CAF concentration using the Western blot technique. All participants were supplemented orally with vitamin D3 before the training intervention period commenced. Appendicular lean mass (aLM) was evaluated by dual energy X-ray absorptiometry. Multiple linear regression models were used to identify factors significantly associated with CAF concentration. Results: Appendicular lean mass, age and sex were identified as significant explanatory factors for CAF concentration. Gait speed and hand grip strength were not associated with CAF concentration. Male participants showed a strong correlation (r = − 0.524) between CAF serum concentration and aLM, whereas this was not the case (r = −0.219) in females. Vitamin D supplementation and physical exercise were significantly associated with a reduction in CAF concentration, especially in participants with initially high CAF concentrations. Conclusions: C-terminal Agrin Fragment could be a potential marker for identifying sarcopenia in a subgroup of affected individuals in the future. The decline of muscle mass seems to be a CAF-associated process in males, whereas the situation in females may be more complex and multifactorial. CAF concentration is reduced by vitamin D supplementation and physical exercise and therefore suggests a potentially positive effect on NMJs. Further prospective studies of sarcopenic patients in addition to muscle biopsy and electromyographical investigations are planned to verify the external validity of the CAF concept. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Sarcopenia is characterised by the progressive loss of skeletal muscle mass and function, bearing an increased risk of adverse health ⁎ Corresponding author. Tel.: + 49 911 300050; fax: + 49 911 3000525. E-mail address: [email protected] (M. Drey). 1 FiAT intervention group: A. Zecha,b; E Freibergerb; K. Pfeiferb; T. Bertschc. a University of Hamburg, Department of Movement Science, Mollerstrasse 2, 20148 Hamburg, Germany bUniversity of Erlangen-Nuremberg, Institute of Sport Science and Sport, Gebbertstrasse 123b, 91058 Erlangen, Germany cKlinikum Nürnberg, Institute for Clinical Chemistry, Laboratory Medicine and Transfusion Medicine, Prof. Ernst Nathan Strasse 1, 90419 Nuremberg, Germany. 0531-5565/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2012.05.021

outcomes such as physical disability, poor quality of life and death (Cruz-Jentoft et al., 2010). The mechanisms behind this age-associated phenomenon have not been completely elucidated. A multidimensional aetiological concept of sarcopenia is currently under discussion. This paper focuses on age-associated changes at the neuromuscular junction (NMJ), which may be of major importance for the development of sarcopenia (Edström et al., 2007, Jang and Van Remmen, 2011). Agrin, an extracellular proteoglycan, is synthesised in motoneurons, transported along the axons and finally released into synaptic basal lamina, where it induces postsynaptic differentiation including acetylcholine receptor (AChR) clustering. Agrin is therefore essential

M. Drey et al. / Experimental Gerontology 48 (2013) 76–80

for the formation and stabilisation of NMJs (Wu et al., 2010). Agrin is inactivated by cleavage from neurotrypsin, a synaptic protease, which frees a soluble 22 kDa C-terminal Agrin fragment (CAF) that can be detected in human serum (Stephan et al., 2008, Frischknecht et al., 2008, Bolliger et al., 2010). Experiments with transgenic mice overexpressing neurotrypsin in spinal motoneurons have shown the full sarcopenia phenotype, including a reduced number of muscle fibres, increased heterogeneity of fibre thickness, more centralised nuclei, fibre-type grouping and an increased proportion of type I fibres (Bütikofer et al., 2011). Thus elevated levels of CAF should be indicative of sarcopenia caused by degeneration of NMJs. The aim of the present study is to evaluate relevant influencing variables on the concentration of cleaved C-terminal Agrin Fragments in the serum of prefrail community-dwelling older adults. Furthermore, the effects on CAF concentration by vitamin D supplementation and physical exercise are investigated.

77

2.2. Detection of CAF in serum samples Participant's serum samples were stored in 2 ml screw-cap polypropylene tubes and frozen at −80 °C. The CAF concentration was determined as described in Hettwer et al. (2012). Briefly, CAF concentrations were measured by using known concentrations of recombinantly produced CAF by Western blotting. 2.3. Body composition Body composition was assessed using a dual energy X-ray absorptiometry (DXA) scanner (Lunar Prodigy, GE Healthcare Technologies, USA). Appendicular lean mass (aLM) was calculated as the sum of the lean mass of both arms and legs. For aLM, Cronbachs alpha for repeated measures on a sub-sample of 20 participants was 0.998. 2.4. Statistical analysis

2. Material and methods 2.1. Participants Participants were drawn from a randomised, controlled, singleblinded 12-week intervention study that investigated the effects of power training versus strength training on physical performance in prefrail community-dwelling older adults. The detailed description of the participants, intervention programme and results was published recently (Drey et al., 2011). Briefly, the 69 prefrail participants, aged between 65 and 94 years, were randomised into three arms: 1. “power training” (n = 24), 2. “strength training” (n = 23) and 3. “control group” (n = 22). The time line of the trial is shown in Fig. 1. Before the intervention period commenced, all 69 participants began supplementation with vitamin D3 up to 2000 IU per day for two months, because randomised controlled trials in older adults investigating the effects of vitamin D substitution on physical performance show a clear tendency towards the improvement in muscle strength and physical performance due to this intervention (DirksNaylor and Lennon-Edwards, 2011). For the inclusion criterion “prefrailty”, the definition given by Fried and colleagues was applied (Fried et al., 2001). Power training is a specific modification of strength training where the concentric part (lifting or pushing) is completed as quickly as possible, whereas the eccentric part (lowering) should be completed in approximately 2–3 s. In strength training, both movements were performed in approximately 2–3 s. Both exercise modes resulted in significant improvements of physical performance with no statistical difference between the two modes. To measure CAF concentrations, frozen sera from the abovementioned study participants were used. The study was approved by the Medical Ethics Committee of the local university and registered at clinicaltrials.gov under NCT00783159.

2.4.1. Baseline data analysis Baseline data analysis is performed as a cross-sectional investigation at T0. The participants' characteristics are shown as means with accompanying standard deviation. Scatter plots between CAF concentration and aLM and CAF concentration and age for both sexes are shown. The degree of correlation was quantified by Pearson's correlation coefficient. To identify the association between CAF concentration and aLM, adjusted for potential confounders, multiple linear regression models were used. In addition to variables with potential influence on lean mass (sex and age), parameters of physical performance (hand grip strength and gait speed) from the European consensus definition of sarcopenia (Cruz-Jentoft et al., 2010) were included in the model. Based on the full model, non-significant variables were excluded in a step-wise backward selection approach. 2.4.2. Intervention data analysis The same models were used to identify potential explanatory factors for changes in CAF concentration during vitamin D supplementation (CAF concentration at T0–CAF concentration at T1) and physical exercise (CAF concentration at T1–CAF concentration at T2). The control group was not considered in the analysis of the effect of exercising. In addition to aLM, sex and age, the variable containing the difference in 25OHD3 concentration at T0 and T1 (VitD (T0–T1)) is included in the model investigating the vitamin D effect. Based on the CAF cut-off value of 4.66 ng/ml (Hettwer et al., 2012), values for CAF concentrations at T0 and T1 were categorised (CAFcat (T0), CAFcat (T1)) and added to each model. To investigate the mode of training, the variable mode (strength training vs. power training) was added. Finally, the remaining significant variables were tested using an unpaired t-test to show mean differences in CAF concentrations. The level of significance was set at 5%; all tests were two-sided. In view of the exploratory nature of statistical analyses, no alphaadjustment techniques were employed. The statistical analysis was performed using PASW 19.0 (IBM-SPSS Inc., Chicago, Il, USA). 3. Results 3.1. Baseline data analysis (T0)

Fig. 1. Study timeline. Bold boxes indicate the groups investigated during vitamin D supplementation and exercise.

The characteristics of prefrail community-dwelling older adults are shown in Table 1. Table 2 shows the standardised regression coefficients of the multiple linear regression models at baseline (T0). The dependent variable for all models is CAF concentration at T0. Full model 1 contains all variables. The following models 2 and 3 are reduced step-wise for the non-significant variables gait speed and hand grip strength, respectively. Thereby, model 3 consists of the three significant remaining variables: aLM, sex and age. Figs. 2 and 3

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M. Drey et al. / Experimental Gerontology 48 (2013) 76–80

CAF in ng/ml

Table 1 Characteristics of prefrail community-dwelling older adults.

Age [y] BMI [kg/m²] CAF [ng/ml] aLM [kg]

x male (r= -0.524*)

Male (n = 21)

Female (n = 47)

p-Value

79.4 27.8 5.4 21.9

76.0 29.0 4.5 16.4

0.053 0.266 0.145 b 0.001

(7.6) (3.5) (2.9) (3.4)

(6.1) (4.4) (2.2) (1.9)

Values are expressed as: mean (SD). P-values indicate differences between both sexes (t-test).

female (r= -0.219) 14

12

10

8

show the scatter plot between CAF concentration and aLM (male: r = −0.524, p = 0.015; female: r = −0.219, p = 0.140) and CAF concentration and age (male: r = 0.349, p = 0.121; female: r = 0.331, p = 0.023) for both sexes respectively.

6

4

3.2. Vitamin D supplementation (T0–T1) 2

Table 3 shows the standardised regression coefficients of the multiple linear regression models for vitamin D supplementation (T0– T1). The dependent variable for all models is the difference in CAF concentration at T0 and T1. In this model, only the CAF concentration at T0 is significantly associated with the change in CAF concentration during vitamin D supplementation. A significant reduction of CAF concentration in the group with initially high CAF concentrations was found (Table 4). 3.3. Physical exercise (T1–T2) Table 5 shows the standardised regression coefficients of the multiple linear regression models for the physical exercise period (T1– T2). The dependent variable for all models is the difference in CAF concentration at T1 and T2. In this model only CAF concentration at T1 is significantly associated with the subsequent change in CAF concentration during physical exercise. The unpaired t-test (Table 4) depicts a significant reduction in CAF concentration in the group with initially high CAF concentrations. The training mode itself merely shows the influence by trend (p = 0.077 ≤ 2α) on the change in CAF concentrations. Further analysis exhibits by trend (p = 0.089 ≤ 2α, not shown here) that the reduction in CAF concentration is stronger in the power training group than in the strength training group.

0

aLM in kg 10

15

20

25

30

35

*) p = 0.015 Fig. 2. Scatter plot between CAF concentration and aLM at T0 for both sexes.

further significant explanatory factors of CAF concentration. Correlation analysis shows a stronger association in males than in females, suggesting a gender effect. Vitamin D and physical exercise seem to counteract the process of degeneration at the NMJs by reducing the concentration of CAF. The role of neurodegenerative mechanisms in the aetiology of sarcopenia has not yet been examined thoroughly. It is known from embryonic studies that the development of neuromuscular synapses is a highly controlled developmental event. The current mindset is that NMJs are formed through a dialogue between muscle and motor neurons. In this concept, muscle initiates the zone where NMJs will be

CAF in ng/ml

x male (r= 0.349) female (r= 0.331*)

4. Discussion 14

The present study focused on the degeneration of NMJs, which may be relevant in the multifactorial aetiology of sarcopenia. The post-hoc evaluation of CAF concentration as a marker for loss of muscle mass caused by degeneration of NMJs in older adults identified sex and age as

Table 2 Standardized regression coefficients of the multiple linear regression models investigating the baseline data (T0).

12

10

8

6 Model

Standardised regression coefficients

p-Value

R²

1

− 0.498 0.427 0.231 0.109 − 0.071 − 0.474 0.417 0.263 0.087 − 0.422 0.428 0.254

0.015 0.019 0.093 0.501 0.588 0.017 0.021 0.033 0.577 0.015 0.017 0.036

0.227

2

3

aLM Sex Age Hand grip strength Gait speed aLM Sex Age Hand grip strength aLM Sex Age

n = 68, dependent variable: CAF concentration at T0.

4

2 0.223

0

age in y 0.219

65

70

75

80

85

90

95

*) p = 0.023 Fig. 3. Scatter plot between CAF concentration and age at T0 for both sexes.

M. Drey et al. / Experimental Gerontology 48 (2013) 76–80 Table 3 Standardised regression coefficients of the multiple linear regression models investigating the effect of vitamin D supplementation (T0–T1).

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Table 5 Standardised regression coefficients of the multiple linear regression models investigating the training effect.

Model

Standardised regression coefficients

p-Value

R²

Model

Standardised regression coefficients

p-Value

R²

1

− 0.258 0.345 0.033 0.028 0.394 − 0.257 0.350 0.033 0.400 − 0.266 0.363 0.411 0.167 0.463 0.473

0.132 0.050 0.791 0.806 0.002 0.130 0.044 0.793 0.001 0.107 0.028 b0.001 0.129 b0.001 b0.001

0.283

1

− 0.051 0.172 − 0.082 0.452 0.248 0.133 − 0.080 0.455 0.255 0.128 0.433 0.254 0.428 0.251

0.821 0.443 0.584 0.004 0.095 0.349 0.585 0.003 0.077 0.361 0.003 0.075 0.004 0.077

0.177

2

3

4 5

aLM Sex Age VitD (T0-T1) CAFcat (T0) aLM Sex Age CAFcat (T0) aLM Sex CAFcat (T0) Sex CAFcat (T0) CAFcat (T0)

2

0.236

0.247

3

4

0.228 0.211

aLM Sex Age CAFcat Mode Sex Age CAFcat Mode Sex CAFcat Mode CAFcat Mode

(T1)

(T1)

(T1) (T1)

0.198

0.212

0.215

n = 42 (control group is not considered), dependent variable: CAF concentration at T1– CAF concentration at T2, CAFcat (T1): categorised CAF concentration at T1 regarding cut-off of 4.66 ng/ml, mode: training mode (strength vs. power).

n = 68, dependent variable: CAF concentration at T0–CAF concentration at T1, CAFcat (T0): categorised CAF concentration at T0 regarding cut-off of 4.66ng/ml, VitD (T0– T1): difference in 25OHD3 concentration at T0–difference in 25OHD3 concentration at T0.

to some extent for the loss of muscle fibres, resulting in adequate muscular performance (Deschenes et al., 2010, Aagaard et al., 2010). In the present study it was found that vitamin D supplementation significantly reduces CAF concentrations. This was especially the case for participants with initially high CAF levels. A decline in CAF concentrations implies a reduction in neurotrypsin activity. At present, little is known about the regulation of transcription of the neurotrypsin gene; a 40% down regulation has been reported by administration of ethynylestradiol but not tamoxifen to ovariectomised mice (Fong et al., 2010). A direct regulation of neurotrypsin transcription by vitamin D seems unlikely since the multiple linear regression models were unable to find a direct correlation between changes in vitamin D blood levels and the reduction of CAF concentrations. In addition, neither neurotrypsin nor agrin has been identified as vitamin D regulated genes (Ramagopalan et al., 2010). It seems that the modulating effect of vitamin D on CAF is indirect and only apparent in subjects with elevated CAF levels. This reduction is also limited and does not decrease CAF levels to normal levels. Therefore further inhibition of neurotrypsin may still be necessary by a small molecule inhibitor (Hettwer et al., 2012). Physical exercise in the study population described here is associated with a significant reduction in CAF concentration, especially in participants with initially high CAF concentrations. Furthermore, our data suggest that especially power training, involving high neuromuscular activity, is responsible for the reduction of CAF concentrations. In line with the multifactorial aetiology of sarcopenia, loss of muscle mass is caused to some extent by a decrease in the number of fast motoneurons innervating type II fibres (McNeil et al., 2005). Apparently, there is a continual process of denervation and reinnervation of skeletal muscle fibres that leads to the death of some fibres, whilst others are re-innervated by slow motoneurons, resulting in a conversion of the related fibre to a type I fibre (Deschenes et al., 2010). Investigations into this re-innervating

formed and neurons strengthen the newly formed contacts by the secretion of agrin (Lin et al., 2008). The extracellular proteoglycan agrin, which is synthesised in motoneurons, transported along axons and finally released into synaptic basal lamina, induces postsynaptic differentiation including AChR clustering (Wu et al., 2010). It is inactivated by cleavage from neurotrypsin, a synaptic protease which frees the soluble 22 kDa CAF that can be detected in human serum (Stephan et al., 2008, Frischknecht et al., 2008). As a consequence, the Cterminal moiety of agrin disappears from the NMJ and leaves the NMJ unprotected, ending in a deterioration of the endplate. The mouse model overexpressing neurotrypsin in motoneurons showed fragmentation and disassembly of NMJs, with its full phenotype of sarcopenia (Bütikofer et al., 2011). These observations seem to qualify CAF as a marker for sarcopenia caused by degeneration of NMJs. In the present study a post-hoc analysis of CAF serum concentration in prefrail community-dwelling older adults demonstrated a linear negative age-associated correlation with appendicular lean mass in male participants. Observations from the animal model showing that inactivated agrin with consecutively high CAF concentrations were responsible for the deterioration of the endplate, resulting in muscle atrophy, are in accordance with the observation in the present study. Considering the multifactorial aetiology of sarcopenia, the high correlation in males suggests that the decline in muscle mass is a strongly CAF-dependent process, whereas the situation in female participants may be more complex and multifactorial. This is an interesting finding that points towards significant gender differences in the aetiology of sarcopenia. Thus, CAF seems to be a marker for identifying a subgroup of sarcopenic patients caused by degeneration of NMJs. In the present study no parameter of physical function (gait speed) and muscle strength (hand grip strength) could be identified as a significant explanatory factor for CAF concentration. One explanation for this finding could be that nerval sprouting compensated

Table 4 Effects on CAF during vitamin D supplementation (T0–T1) and exercise (T1–T2) in low versus high CAF groups.

Vitamin D Exercise

Low CAF High CAF Low CAF High CAF

T0

T1

3.32 (0.71), 42 7.09 (2.47), 26 –

3.43 5.29 3.14 5.69

(0.97), (1.72), (0.84), (0.88),

42 26 30 16

T2

T0–T1

T1–T2

p-Value

–

− 0.11 (1.00), 42 1.80 (2.58), 26 –

–

b 0.001

3.40 (0.84), 28 4.78 (1.77), 14

− 0.28 (1.04), 28 0.93 (1.50), 14

CAF values are expressed as: mean (SD), n. T0: baseline, T1: end of vitamin D supplementation and start of training, respectively, T2: end of training. P-values (t-test) indicate differences between low and high CAF groups during vitamin D supplementation (T0–T1) and during exercise (T1–T2).

0.004

80

M. Drey et al. / Experimental Gerontology 48 (2013) 76–80

process have shown that both chronic inactivity and neuromuscular hyperactivity, such as excessive endurance training, decrease the muscle fibre re-innervation capacity (Tam and Gordon, 2003). As a consequence, strength training, and especially power training involving high neuromuscular activity for a short time, are considered to be an intervention with high re-innervating potential (Aagaard et al., 2010). The tendency towards a reduction of CAF concentrations associated with power training in the present study is evaluated as an indication of this argumentation. The fact that this effect could not achieve statistical significance was mainly due to the low level of power. These findings are in accordance with the findings of Deschenes et al. (2010) that a high amount of neuromuscular activity protects against degeneration at NMJs resulting in sarcopenia. In our study only participants with initially high CAF concentrations significantly reduced their CAF levels, although participants in both groups benefited from the training. This observation indicates that other mechanisms, such as neural adaptations, increased voluntary activation of agonists or reductions in antagonist co-activation, lead to training-induced improvements. 5. Conclusions The C-terminal Agrin Fragment caused by degeneration of the neuromuscular junction can be measured in human serum. CAF appears to be a promising marker for a subgroup of sarcopenic patients caused by degeneration of the neuromuscular junction. Vitamin D supplementation and physical exercise reduce CAF concentration and therefore suggest a potentially positive effect on NMJs. To verify the external validity of the CAF concept, additional prospective studies in sarcopenic individuals that include muscle biopsy and electromyographical testing are being planned. Acknowledgement Michael Drey and Jürgen M. Bauer were supported by a Forschungskolleg Geriatrie Grant from the Robert Bosch Foundation, Stuttgart, Germany. References Aagaard, P., Suetta, C., Caserotti, P., Magnusson, S.P., Kjaer, M., 2010. Role of the nervous system in sarcopenia and muscle atrophy with aging: strength training as a countermeasure. Scand. J. Med. Sci. Sports 20, 49–64. Bolliger, M.F., Zurlinden, A., Lüscher, D., Bütikofer, L., Shakhova, O., Francolini, M., Kozlov, S.V., Cinelli, P., Stephan, A., Kistler, A.D., Rülicke, T., Pelczar, P.,

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