Örebro University
School of Health and Medical Sciences
Master program: Sports Physiology and Medicine, 120 credits Degree project, 45 credits, Spring 2012
Serum Hs-CRP in elderly women affects the proliferative
capacity of human myoblast
Author: Jenny Ewen
Supervisor: Fawzi Kadi
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Abstract
Introduction
Chronic low-grade inflammation is believed to affect muscle regeneration and to contribute to an age-related decline in muscle mass. Levels of circulating cytokines are negatively age-related to muscle mass in elderly. The aim of this study is to investigate the effect of serum of elderly women with different levels of the inflammatory marker C-reactive protein on the proliferative capacity of human myoblasts. A pilot study was also preformed to investigate a possible effect of resistance training on human myoblast proliferative capacity.
Material and Methods
16 healthy women (age 65-70 yrs) performed a health assessment with blood samples taken and analyzed for Hs-CRP level. Physical activity level was assessed using accelerometry. Myoblasts were extracted from human muscle biopsy samples from vastus lateralis. 30 000 cells was seeded in 35 mm collagen-coated dish at day 1 (0 hour) and cultured in proliferation media with DMEM, 15% human serum and 2% Ultroser G. At day 4 (96h) the cells were counted for evaluation of population doubling time. Human myoblasts were incubated with serums from each of the 16 women. A pilot study was performed to examine the effects of resistance training on the proliferative capacity of human myoblasts. This was done by using serum from one subject who participated to a supervised resistance training program for 24 weeks. Blood sampling was performed before and after the intervention.
Results
Significant associations were found between myoblast DT and serum Hs-CRP level. A relationship was also demonstrated between serum Hs-CRP level and BMI. Using a linear regression analysis it was found that serum Hs-CRP level explained 45 % (p=0.004) of the variability in myoblast DT. A pilot study showed increased strength in 10RM leg extension and leg press with 22% and 46% respectively. The proliferative capacity of myoblast cultured in serum obtained after the training period increased with 2 %. Conclusion
Data from the present study suggest that systemic factors in the biological environment such as the level of inflammation markers (Hs-CRP level) in serum affect the proliferative capacity of muscle cells in old women negatively.
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Introduction
The population of elderly in the western world is increasing steadily. Aging is accompanied by a number of clinical conditions (de Groot et al, 2004) and it also affects the skeletal muscle. With an increase in inflammatory biomarkers and an impaired regenerative capacity (Woods et al 2012; Renault et al, 2002) aging is associated with a slow and progressive loss of muscle mass, strength and power resulting in muscle weakness (Frontera et al, 2000; Kamel, 2003; Janssen et al, 2002). This age-related biological change can lead to pathological conditions negatively affecting independence and quality of life among the elderly (de Groot et al, 2004; Janssen et al, 2002). Although the knowledge about the effects of aging has increased, aging remains one of the most poorly understood biological phenomena, due to its complexity and the difficulty to separate the effects of normal healthy aging and those manifested as a consequence of age-related disease. In addition to the cellular senescence theory of aging in which the cellular signal responses to stress and damage are thought to become impaired with aging (Fossel, 2000), the inflammatory theory of aging, where the activation of redox -sensitive transcriptional factors causes an up-regulation of pro-inflammatory gene expression in elderly has gained more attention lately. This increased level of inflammatory genes during aging has been linked to a number of related diseases (Giunta, 2006) and it is hypothesized that it is related to the age-associated decrease in muscle function.
Age related low- grade inflammation
During aging there is a decline in the immune system function promoting age-associated slightly elevated circulating levels of inflammatory markers such as TNF-alpha and interleukin-6 (IL-6) compared to young (Wei et al, 1992: Chung et al 2009). A slightly elevated levels of circulating cytokines is a strong risk factor of morbidity and mortality in the elderly (Woods et al, 2012), with an increased risk of developing cardiovascular disease, cancer and diabetes (Bruunsgaard & Pedersen, 2003; Chung et al 2009). Although increased levels of inflammatory markers is often a result of age- related disease (Chung et al 2009), increased levels of inflammatory markers have also been seen in healthy elderly (Wei et al 1992; Bruunsgaars & Pedersen, 2003).
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Low-grade systemic inflammation seen in elderly is defined as a two to four fold elevation in circulating levels of cytokines as well as acute phase protein such as C – reactive protein (CRP) compared to young (Chung et al, 2009; Woods et al, 2012). In healthy young adults the median concentration of CRP in the blood is 0.8 mg/l (Pepys and Gideon, 2003). The physiological role of CRP is to bind on phosphocholine on bacteria and damaged cells and to participate in the clearance of apoptotic cells. The circulating value of CRP reflects ongoing inflammation most accurately and is a useful nonspecific biochemical marker of inflammation, thus CRP is not affected by eating or drugs unless they also affect the underlying reason to the acute-phase stimulus (Pepys and Gideon, 2003). Whereas a CRP-test is a general test to check for inflammation in the body, a more sensitive test called high sensitive CRP (Hs-CRP) test has become a common clinical test to identify risks of cardiovascular disease. Levels higher than 3.0 mg/l is considered high risk for cardiovascular disease.
While inflammation is necessary to recover from injury and infection, failure to completely develop an immune response due to the decline in the immune system with increasing age, could be a contributing factor to the chronic state of low-grade inflammation associated with age (Woods et al, 2012; Degens, 2010). Except the decline in the immune system a wide range of environmental factors, including smoking, presence of sub-clinical infections, obesity and genetic factors has been investigated and has been show to correlate with higher levels of circulating cytokines (Bruunsgaard & Pedersen, 2003; Chakravarty et al 2012). Lower inflammatory biomarker concentrations have been reported in individuals who reported frequent physical activity (Colbert et al, 2004; Elosua et al, 2005). As little as 1 day/week of physical activity are associated with lower CRP levels compared with sedentary individuals. In addition, more frequent physical activity (>4 days/week) is associated with additional reductions in CRP levels (Plaisance & Grandjean, 2006). While frequent physical activity (Colbert et al, 2004; Elosua et al, 2005) and training (Donges et al 2010; Ogawa et al, 2010) has been shown to reduce CRP levels in older adults, this effect has also been shown to be partially mediated by a reduction in trunk fat (Vieira et al, 2009).
There are indications of chronic low-grade inflammation affecting muscle regeneration and contributing to the age-related decline in muscle mass and function. While, high levels of TNF-alpha and IL-6 are negatively related to muscle mass and strength in elderly, it is hypothesized
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that low-grade inflammation over many years is believed to delay differentiation of satellite cells and hamper maintenance of the skeletal muscle, resulting in progressive loss of skeletal muscle (Degens, 2007; Visser et al, 2002; Chung et al, 2009).
Muscle growth and regeneration
Although skeletal muscle is mainly a post-mitotic tissue it also processes a population of cells referred to as satellite cells. The skeletal muscle has a remarkable capacity to regenerate in response to injury. Successful regeneration, growth and maintenance of skeletal muscle require the recruitment of satellite cells (Charge & Rudnicki, 2004). Satellite cells are located between the sarcolemma and the basal lamina of the myofibers and fitfulfill the criteria for a stem cell. Satellite cells divide, fuse with the myofiber or form myotubes to repair damage and to generate new muscle tissue (Zammit et al, 2006). Satellite cells are called myoblast as soon as they are activated and leave their location underneath the basal lamina of the muscle fiber. When satellite cells are activated they proliferate with an upregulated of myogenic markers (Bigot et al, 2008; Charge & Rudnicki, 2004; Zammit et al 2006).
The numbers of satellite cells available will determine the regenerative potential of the muscle. As the pool of satellite cells will decrease with age (Carlson et al, 2009; Kadi et al 2004), ageing is believed to negatively affect the regenerative properties of the skeletal muscle (Renault et al, 2000; Conboy et al, 2005; Pietrangelo et al, 2009) with a potentially loss of muscle mass and function referred to as sarcopenia (Kamel, 2003). The regenerative capacity of skeletal muscle is linked to the number of satellite cells but also to their activation, proliferation and differentiation capacity (Zammit et al, 2006; Renault et al, 2000; Carlson et al, 2009). Renault and coworkers (2000) demonstrated a limited life span of human satellite cells in vitro related to the age of the donor and that the number of non- or slow-dividing satellite cells increases with donor age, leading to the suggestion that sarcopenia might be a result of a decreased proliferative capacity of aged satellite cells.
The hypothesis stating that systemic factors in the biological environment of the muscle affect muscle regeneration was raised when an aged rat muscle successfully regenerated when
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implanted into a young rat host (Carlson & Faulkner, 1989). This hypothesis was further supported when the decrease of muscle regenerative potential with aging was reversed through the modulation of systemic factors (Conboy et al, 2005): using a parabiotic model, aged muscle was rejuvenated when old animals shared circulation with young animals. (Conboy et al, 2005). The proliferative capacity was impaired when mouse cells were cultured in serum from old mice (Carlson & Conboy, 2007). Additionally, an improved regenerative response was reported in aged human myoblasts cultured in young human serum (Carlson and al, 2009). These results were not confirmed in a subsequent study (George et al, 2010). The reason for this discrepancy remains unknown. In both studies no measurement of serum inflammatory markers was performed.
As aging is associated with a slow and progressive loss of muscle function resulting in muscle weakness (Frontera et al, 2000; Kamel, 2003; Janssen et al, 2002) resistance training has been proven effective in maintaining and restoring muscular strength capacity among the elderly (Peterson et al, 2010; Vincent et al, 2002). In prevention of sarcopenia resistance training is believed not only to result in an increased muscular strength capacity but also contribute to better health in older age. The effects of resistance training on the proliferative capacity of myoblasts isolated in elderly subjects are however still unknown.
The aim of this study is to investigate the effect of elderly woman serum Hs-CRP level on the proliferative capacity of human myoblast. Furthermore, a pilot study was conducted in order to determine if resistance training in elderly can affect human myoblast proliferative capacity.
Method
Subjects and ethics
16 healthy women in the age of 65-70 years were recruited with the help from local media. All subjects performed a health assessment including measured weight and height. Blood samples were taken and analyzed for High sensitive C – reactive protein (Hs-CRP) level. A six day physical activity assessment with accelerometers was conducted to investigate current physical activity patterns. Subjects’ characteristics are presented in table 1. The study complies with the
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standards set by the Declaration of Helsinki and all subjects were carefully informed about the possible risks and benefits of the study. All subjects gave their written informed consent before entering the study. An application was sent to the central ethical review board of Uppsala. The study design was approved by the ethical committee (DNR: 2011/033).
Table 1 Subject characteristics. Data presented as mean ± SD
Laboratory measurements
Physical Activation registrationPhysical activity (PA) was measured for all subjects. PA was assessed with an Actigraph accelerometer set at 60 second epochs. The Actigraph accelerometer measures vertical acceleration as counts providing an indication of the intensity of physical activity (Hagströmer et al, 2010). The subjects were instructed to wear the accelerometer on the hip using an elastic belt and to remove the device while sleeping, swimming and showering. The subjects were also instructed to note if they used a bike for transport with the duration of the bike ride. Final PA count value was calculated as a mean count of a total of 6 days.
Blood collection and analysis
Blood samples were collected in the morning after an overnight fast. Blood was drawn from a forearm vein into sterile vacutainer tubes. Hs-CRP was analyzed using Advia 1800 (Siemens) at the department of clinical chemistry Örebro university hospital.
N Age Weight (kg) Height (cm) Hs-CRP (mg/l)
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Anthropometry
Height was measured using a stadiometer. Weight was measured with foot-to-foot bioelectrical impedance analysis (BIA) in the morning after one night fasting. The subjects were wearing underwear standing erect with bare feet on the footpads of the BIA scale. BIA was performed on a Tanita leg-to-leg Body Composition Analyzer (Tanita BCC-420MA, Japan).
Cell culture
Preparation of the cells
Biopsies were obtained from the vastus lateralis muscle of a healthy woman age 65-70 years. Under sterile conditions the tissue was minced with a scalpel on a microscope slides (Thermo scientific). The pieces were placed in freezing media (90% Fetal bovine Serum, 10% DMSO) and frozen progressively at 4°C, -20°C, -80°C and finally stored in liquid nitrogen until cultured.
Migration from muscle tissue
The biopsy pieces were quickly thawed in 37°C and rinsed carefully with medium with DMEM and HEPES. Explants were trapped inside a thin layer of Matrigel (BD Matrigel Matrix EHS free from BD Biosciences) in 35 mm collagen-coated dishes with medium (DMEM/HEPES) added on top of the layer. The explants were cultured for 8 days with medium changed every 2 days. After 8 days the migrating cells were enzymatically harvested using Dispase (BD Biosciences) and sub cultured in proliferating media. At 80% confluence harvested cells were purified with an immune-magnetic sorting system (Miltenyi Biotec, USA) (Barro et al 2010; Kitzmann et al, 2006).
Purified myoblast were planted in collagen-coated dishes and cultured in proliferation media composed of DMEM supplemented with 20% fetal bovine serum and 2 % Ultroser G (PALL, life science) at 37°C in humidified atmosphere with 5% CO2. At cell purification, all human
myoblasts were considered to be at P1. All experiments were performed at P4, to be as close to original proliferation as possible and to without doubt avoid premature replicative senescence which interferes with the proliferation and differentiation process.
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Population doubling time (Myoblast DT)
Counting was performed using a hemocytometer. The hemocytometer was loaded twice with 10µl and an average of 5 squares was counted to get the amount of cells/0.1µl. The counted amount of cells is then multiplied to get the total number of cells according to the formula: total cells = x/ml x sample size. The loaded sample with cells was counted twice to ensure correct counting. Myoblast population doubling time (DT) was calculated at 96 hours according to the formula: MYOBLAST DT = 96/(1n(y/x)/in2), where x is the number of cells initially seeded at day 1 (0h) and y the total number of cells counted at day 4 (96h).
Preliminary tests
Experiments were performed investigating the possibility of working with smaller amounts of medium and cells. A test was performed to determine the possibility of proliferation in a 24-well plate. The aim was to investigate if 1ml or 0,5ml is the proper quantity of media in the well for proliferation based on Myoblast DT. Fetal bovine serum at 20 % was used in the media. During proliferation to confluence the proliferating media is normally changed every two days. Therefore, the experiment also aimed to investigate the possibility to grow the cells without changing the medium during the 96h of proliferation. The wells were seeded with 8000 cells and were counted for Myoblast DT at 96h. Duplicates of every well were conducted. We also compared the total amount of cells is a 35 mm and a 24-well dish. Our results confirm the possibility of proliferating cells in a 24-well plate with 1 ml of media without media change during 96h (table 2). However, the total number of cells generated at 96h in a 24-well plate was well below that obtained with a 35 mm dish. Therefore, we performed our experiments in a 35mm dish.
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Table 2 Doubling time of myoblast under different culture conditions in a 24-well
Seeded cells
Amount of
Proliferation media Change of media Myoblast DT (h)
8000 1ml No 27,9
8000 1ml Yes 37,6
8000 500µl No 88,3
8000 500µl Yes 46,9
Tests were also performed to determine the appropriate concentration of human serum to use in proliferation media for future experiments. A test was performed in a 24-well plate, with 1 ml of proliferation media, proliferating for 96 hours. The media was not changed during the 96 h of proliferation. Myoblast were cultured in human serum concentration of 5, 10 and 20% (table 3). Control wells with proliferation media with 20 % fetal bovine serum were used resulting in a Myoblast DT of 35 h. The wells were seeded with 8000 cells and were counted for Myoblast DT at 96h. Based on these experiments the percentage of human serum in proliferating media was optimal at 15 %.
Table 3 Concentration of human serum used in proliferation media and doubling time of myoblasts
% of human serum Myoblast DT (h)
5 72,6
10 44.6
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Effect of serum Hs-CRP concentration on the proliferative capacity of human myoblast
In order to study the effect of inflammation on proliferative capacity of skeletal muscle, human myoblast from old women were incubated with 15 % serum from each one of the 16 subjects. Based on preliminary tests 30 000 cells at P4 was seeded in 35 mm collagen-coated dish at day 1 (0 hour) and cultured in 2ml proliferation media with DMEM, 15% subject human serum and 2% Ultroser G. The proliferating media was not changed during the four days of culture. At day 4 (96h) the cells were trypsinized, centrifuged for 5 min at 1500 rpm and resuspended in 1 ml of DMEM before counting for Myoblast DT.
Myoblasts were cultured in a 35 mm collagen-coated dish with 15% fetal bovine serum and one with 15% young human serum as controls.
Pilot study: The effects of training on myoblast proliferation capacity
A pilot study was performed to examine the effects of resistance training on the proliferative capacity of human myoblasts. This was done by using serum from one subject who participated to a supervised resistance training program for 24 weeks (table 4). Blood sampling was
performed before and after the intervention. This pilot study was conducted within the context of a larger research project where 60 women participated to a randomized-controlled trial on the effects of resistance training on health.
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Table 4 characteristics of the subject who participated in a resistance training program for 6
months Pre Post Age (yrs) 67 67 Height (cm) 170 170 Weight (kg) 83 83,4 Fat % 41,9 41,5 FFM (kg) 48,2 48,8 10 RM leg extension (kg) 35 45 10 RM leg press (kg) 70 125 Training program
24 weeks progressive resistance training program for the upper and lower extremities with the goal to increase muscle mass and strength was performed. Weeks 1-2 consisted of a familiarization period where the subject trained at an intensity of 50% of 1 repetition maximum. From week 3-24 subjects trained at 10 repetitions maximum (10RM). Prior to intervention start 10RM was tested to ensure correct training intensity. 10RM was then tested regularly to ensure the correct intensity throughout the intervention period. Training consisted of two sessions per week of 3 sets of squats in the Smith machine, leg extension and 2 sets on the leg press. Upper body exercises consisted of seated row, pull down and core stability. At week 12 squat jumps were included at the 1st training session of the week and drop jumps were included at the 2nd session. A five minute warm up on the stepper was performed prior to each training session. The Subject was instructed to follow the BORG scale at intensity 11-13. After each training session the subject performed a 5 minute stretch program.
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Effects of resistance training on the proliferative capacity of human myoblast
In order to investigate the effects of resistance training on the proliferative capacity of human myoblasts, 30 000 human myoblasts at P4 from one subject (table 4) were seeded in 35 mm collagen-coated dish and cultured with proliferation media consisted of DMEM, 2% of ultroser G and 15% of the subject serum pre and post 24 weeks of resistance training. The proliferating media was not changed during the four days of culture. At day 4 (96h) the cells were trypsinized, centrifuged for 5 min at 1500 rpm and re-suspended in 1 ml of DMEM before counting myoblast DT.
Statistics
Statistical analysis was performed using IBM SPSS statistics v.20 software. Pearson correlation was used initially to investigate associations between the variables. Linear regression analysis was used with serum Hs-CRP level as explanatory variable and myoblast DT as the dependent variable. All values are expressed as individual data points or as mean values ± SD. Statistical significant was set at p< 0.05.
Results
Pearson correlation analysis demonstrated significant associations between Myoblast DT and serum CRP level (table 5, fig 1.). A relationship was also demonstrated between serum Hs-CRP level and BMI. BMI and PA count were not associated with myoblast DT (table 5).
The effect of serum Hs-CRP level on myoblast DT was further investigated using a linear regression analysis. Serum Hs-CRP level explained 45 % (p=0.004) of the variability in myoblast DT (table 6).
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Table 5 Correlation analysis between Myoblast DT, PA count, BMI and Hs-CRP.
Myoblast DT BMI PAcount Hs-CRP
Myoblast DT 1
BMI 0.302 1
PAcount -0.163 -0.48 1
Hs-CRP 0.672** 0.692** -0.458 1
** = p< 0.01 * = p< 0.05
Abbreviations: PA count = Physical activity count; BMI = body mass index; Myoblast DT = myoblast population doubling time; Hs-CRP = High sensitive C – reactive protein. (N = 16)
Table 6 Linear regression analysis between Myoblast DT and Hs-CRP
Regression Model
Dependent
variable Independent variable
standardized
β coefficient R2 Sig.
1 Myoblast DT Hs-CRP 0,672 0.45 0.004
Abbreviations: R2= proportion of the variation in the response around the mean that can be attributed to terms in the model rather than to random error; PA count = Physical activity count; BMI = body mass index; Myoblast DT = myoblast population doubling time; Hs-CRP = High sensitive C – reactive protein. (N = 16).
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Fig. 1 The relationship between Hs-CRP level and Myoblast DT. R = 0.67, R2 = 0.45.
Pilot study: The effects of training on myoblast proliferative capacity
24 weeks of resistance training resulted in an increased strength in 10RM leg extension and leg press with 22% and 46% respectively. The proliferative capacity of the myoblasts increased with 2 % after 24 weeks of training (table 7).
Table 7 Presenting subject total myoblast count at 96h and myoblast DT
Serum Pre Serum Post
Total myoblast count 297 000 308 000
Myoblast DT (h) 29.03 28.57 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 0 1 2 3 4 5 6 7 8 P op u lati on d ou b lin g t im e (h ) Hs-CRP level (mg/l)
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Discussion
The main finding of the current study is the association between the level of inflammation (CRP) in serum from old women and myoblast DT. Serum levels of CRP explained 45% of the variability in myoblast DT. This finding suggests an impaired human myoblast proliferation capacity in old with increased human serum CRP level. This result is in line with the inflammatory theory of aging, where the activation of redox -sensitive transcriptional factors causes an up-regulation of inflammatory markers and influences physiological functions. As an increased level of inflammatory markers during aging has been linked to a number of age-related diseases (Giunta, 2006) the current study also show a decreased proliferative capacity of human myoblast due to the occurrence of inflammation.
In vitro, a limited life span of human satellite cells has been related to the age of the donor and the number of non- or slow-dividing satellite cells increased with donor age (Renault et al, 2000; Pietrangelo et al, 2009), giving rise to the hypothesis that systemic factors in the biological environment of the muscle affect muscle regeneration. Results from the current study show that serum Hs-CRP level in elderly affects the proliferative capacity of human myoblasts. This knowledge further adds to the theory of sarcopenia, which might be a result of a decreased proliferative capacity due to low-grade inflammation. In line with the present results, TNF-alpha and IL 6 have been shown to be negatively related to muscle mass and strength in elderly (Chung et al, 2009). Elevated levels of TNF-alpha have been linked to decreased force generating capacity in the myofibrillar in mice (Hardin et al 2008). Our results are in line with experiments where addition of TNF-alpha in cultured mice myoblast inhibited their proliferation and differentiation (Langen et al, 2006; Foulstone et al, 2004).
In humans there are somewhat conflicting reports in age-related changes in myoblasts’ behavior cultured in vitro (Carlson et al, 2009; George et al, 2010; Renault et al, 2000; Pietrangelo et al, 2009). As a low-grade elevation in levels of circulating cytokines is a strong risk factor of morbidity and mortality in the elderly (Bruunsgaard & Pedersen, 2003; Woods et al, 2012) the present study shows for the first time that low-grade inflammation is a contributing factor to impaired regenerative capacity with age.
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The previous two studies investigating the effect of serum from elderly on myoblast proliferation used human serum without taking into consideration the level of inflammation (Carlson et al 2009; George et al, 2010). The aim of the present study was not to compare old and young but to investigate if the level of inflammation in human serum from old woman affects the proliferative capacity of human myoblast from old. The current study is the first to investigate the proliferative capacity of human myoblast cultured in human serum from old women with different serum Hs-CRP levels. Established laboratory methods of purifying human myoblast with an immune-magnetic sorting system was used (Barro et al 2010; Kitzmann et al, 2006) since cells obtained from explants are contaminated with non-myogenic cells. In other studies using human serum, the myogenic purity of the human myoblast cultures was monitored using an antibody specific for desmin (George et al, 2010; Carlson et al, 2009). The percentage of human serum in proliferation media used in the present study is in line with George et al 2010 using 15 % of human serum when comparing proliferation capacity of human myoblast cultured in young and old serum. As in our study, George and coworkers (2010) used 15% human serum in the proliferative media based no differences in the percentage of desmin and Ki67 positive cells between medium containing 10 and 15% human serum.
It has been stated that a wide range of environmental factors, including smoking, infections, obesity and genetic factors might be contributing factors to chronic low-grade inflammation seen in healthy elderly (Bruunsgaard & Pedersen, 2003; Chakravarty et al 2012). Lifestyle factors such as physical activity, training and diet have been shown to have a possible effect on inflammation (Donges et al, 2010; Vieira et al, 2009; Valentine et al, 2009; Ogawa et al, 2010). Hs-CRP level was not correlated with PA count. However, due to the low number of subjects, this result does not preclude the existence of a relationship between PA and CRP. Indeed, previous research suggested that inflammatory markers are lower in older adults with higher levels of exercise (Colbert et al, 2004) and that less time spent physical active correlate with higher serum CRP values (Elosua et al, 2005). The present results showed that serum Hs-CRP level was significantly correlated with BMI. A high BMI associated with a high Hs-CRP level has also been confirmed in studies showing strong association between trunk fat and CRP level
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(Taaffe et al, 2000; Valentine et al, 2009; Vieira et al, 2009). High body fat is likely to be associated with high levels of CRP, since about 25 % of the inflammatory marker IL-6 is produced in adipose tissue. As IL-6 is responsible for signaling the liver to secret CRP, a reduction in body fat likely induces lower CRP levels (Plaisance & Grandjean, 2006).
To determine a potential effect of resistance training on myoblast proliferation capacity a pilot study was conducted. After 24 weeks of resistance training an improvement of muscle strength occurred together with a slight increase of 2 % in myoblast DT. The effect of resistance training on the proliferative capacity of human myoblast has never been addressed before. Our result indicates that changes had occurred in serum in response to resistance training. The increase DT is very small and further experiments are needed to address this issue at a larger scale.
Conclusion
This study is the first to investigate the proliferative capacity of human myoblast cultured in human serum from old women with different Hs-CRP levels. Data from the present study suggest that systemic factors in the biological environment such as the level of inflammation markers (Hs-CRP level) in serum negatively affect the proliferative capacity of muscle cells in old woman. This is in line with the hypothesis that chronic low-grade inflammation in elderly negatively affects muscle regeneration and contributes to an age-related decline in muscle mass and function. Further research is needed to investigate the effects of serum CRP on human myoblast ability to differentiate. Further research is also needed to determine the effects of resistance training on human myoblast proliferation and differentiation in vitro.
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References
Barro, M., Carnac, G., Flavier, S., Mercier, J., Vassetzky, Y., & Laoudj-Chenivesse, D. (2010). Myoblasts from affected and non-affected FSHD muscles exhibit morphological
differentiation defects. J Cell Mol Med, 14(1-2), 275-289.
Bigot, A., Jacquemin, V., Debacq-Chainiaux, F., Butler-Browne, G. S., Toussaint, O., Furling, D., & Mouly, V. (2008). Replicative aging down-regulates the myogenic regulatory factors in human myoblasts. Biol Cell, 100(3), 189-199.
Bruunsgaard, H., & Pedersen, B. K. (2003). Age-related inflammatory cytokines and disease.
Immunol Allergy Clin North Am, 23(1), 15-39.
Carlson, B. M., & Faulkner, J. A. (1989). Muscle transplantation between young and old rats: age of host determines recovery. Am J Physiol, 256(6-1), 1262-1266.
Carlson, M. E., & Conboy, I. M. (2007). Loss of stem cell regenerative capacity within aged niches. Aging Cell, 6(3), 371-382.
Carlson, M. E., Suetta, C., Conboy, M. J., Aagaard, P., Mackey, A., Kjaer, M., & Conboy, I. (2009). Molecular aging and rejuvenation of human muscle stem cells. EMBO Mol Med,
1(8-9), 381-391.
Chakravarty, E. F., Hubert, H. B., Krishnan, E., Bruce, B. B., Lingala, V. B., & Fries, J. F. (2012). Lifestyle risk factors predict disability and death in healthy aging adults. Am J
Med, 125(2), 190-197.
Charge, S. B., & Rudnicki, M. A. (2004). Cellular and molecular regulation of muscle regeneration. Physiol Rev, 84(1), 209-238.
Chung, H. Y., Cesari, M., Anton, S., Marzetti, E., Giovannini, S., Seo, A. Y., Leeuwenburgh, C. (2009). Molecular inflammation: underpinnings of aging and age-related diseases. Ageing
Res Rev, 8(1), 18-30.
Colbert, L. H., Visser, M., Simonsick, E. M., Tracy, R. P., Newman, A. B., Kritchevsky, S. B., Harris, T. B. (2004). Physical activity, exercise, and inflammatory markers in older adults: findings from the Health, Aging and Body Composition Study. J Am Geriatr Soc,
52(7), 1098-1104.
Conboy, I. M., Conboy, M. J., Wagers, A. J., Girma, E. R., Weissman, I. L., & Rando, T. A. (2005). Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature, 433(7027), 760-764.
20
de Groot, L. C., Verheijden, M. W., de Henauw, S., Schroll, M., & van Staveren, W. A. (2004). Lifestyle, nutritional status, health, and mortality in elderly people across Europe: a review of the longitudinal results of the SENECA study. J Gerontol A Biol Sci Med Sci,
59(12), 1277-1284.
Degens, H. (2007). Age-related skeletal muscle dysfunction: causes and mechanisms. J
Musculoskelet Neuronal Interact, 7(3), 246-252.
Degens, H. (2010). The role of systemic inflammation in age-related muscle weakness and wasting Scand J Med Sci Sports, 20(1), 28-38.
Donges, C. E., Duffield, R., & Drinkwater, E. J. (2010). Effects of resistance or aerobic exercise training on interleukin-6, C-reactive protein, and body composition. Med Sci Sports
Exerc, 42(2), 304-313.
Elosua, R., Bartali, B., Ordovas, J. M., Corsi, A. M., Lauretani, F., & Ferrucci, L. (2005). Association between physical activity, physical performance, and inflammatory biomarkers in an elderly population. J Gerontol A Biol Sci Med Sci, 60(6), 760-767. Fossel, M. (2000). Cell senescence in human aging: a review of the theory. In Vivo, 14(1), 29-34. Foulstone, E.J., Huser, C., Crown, A.L., Holly, J.M., Stewart, C.E. (2004) Differential signalling
mechanisms predisposing primary human skeletal muscle cells to altered proliferation and differentiation: roles of IGF-I and TNFalpha. Exp Cell Res, 294: 223–235.
Frontera, W. R., Hughes, V. A., Fielding, R. A., Fiatarone, M. A., Evans, W. J., & Roubenoff, R. (2000). Aging of skeletal muscle: a 12-yr longitudinal study. J Appl Physiol, 88(4), 1321-1326.
George, T., Velloso, C. P., Alsharidah, M., Lazarus, N. R., & Harridge, S. D. (2010). Sera from young and older humans equally sustain proliferation and differentiation of human myoblasts. Exp Gerontol, 45(11), 875-881.
Giunta, S. (2006). Is inflammaging an auto[innate]immunity subclinical syndrome? Immun
Ageing, 3, 12.
Hagstromer, M., Troiano, R. P., Sjostrom, M., & Berrigan, D. (2010). Levels and patterns of objectively assessed physical activity--a comparison between Sweden and the United States. Am J Epidemiol, 171(10), 1055-1064.
Hardin, B.J., Campbell, K.S., Smith, J.D., Arbogast, S., Smith, J., Moylan, J.S., Reid, M.B. (2008) TNF-alpha acts via TNFR1 and muscle-derived oxidants to depress myofibrillar force in murine skeletal muscle. J Appl Physiol, 104(3): 694-9.
21
Janssen, I., Heymsfield, S. B., & Ross, R. (2002). Low relative skeletal muscle mass (sarcopenia) in older persons is associated with functional impairment and physical disability. J Am
Geriatr Soc, 50(5), 889-896.
Kadi, F., Charifi, N., Denis, C., Lexell, J., Andersen, J. L., Schjerling, P., Kjaer, M. (2005). The behaviour of satellite cells in response to exercise: what have we learned from human studies? Pflugers Arch, 451(2), 319-327.
Kamel, H. K. (2003). Sarcopenia and aging. Nutr Rev, 61(5-1), 157-167.
Kitzmann, M., Bonnieu, A., Duret, C., Vernus, B., Barro, M., Laoudj-Chenivesse, D., Carnac, G. (2006). Inhibition of Notch signaling induces myotube hypertrophy by recruiting a
subpopulation of reserve cells. J Cell Physiol, 208(3), 538-548.
Langen, R.C., Schols, A.M., Kelders, M.C., van der Velden, J.L., Wouters, E.F., Janssen- Heininger, Y.M. (2006) Muscle wasting and impaired muscle regeneration in a murine model of chronic pulmonary inflammation. Am J Respir Cell Mol Biol, 35: 689–696. Ogawa, K., Sanada, K., Machida, S., Okutsu, M., & Suzuki, K. (2010). Resistance exercise
training-induced muscle hypertrophy was associated with reduction of inflammatory markers in elderly women. Mediators Inflamm, 2010, 171023.
Pepys, M. B., Hirschfield, G. M. (2003). C-reactive protein: a critical update. J Clin Invest,
111(12), 1805-1812.
Peterson, M. D., Rhea, M. R., Sen, A., Gordon, P. M. (2010). Resistance exercise for muscular strength in older adults: a meta-analysis. Ageing Res Rev, 9(3), 226-237.
Pietrangelo, T., Puglielli, C., Mancinelli, R., Beccafico, S., Fano, G., & Fulle, S. (2009). Molecular basis of the myogenic profile of aged human skeletal muscle satellite cells during differentiation. Exp Gerontol, 44(8), 523-531.
Plaisance, E. P., & Grandjean, P. W. (2006). Physical activity and high-sensitivity C-reactive protein. Sports Med, 36(5), 443-458.
Renault, V., Piron-Hamelin, G., Forestier, C., DiDonna, S., Decary, S., Hentati, F., Mouly, V. (2000). Skeletal muscle regeneration and the mitotic clock. Exp Gerontol, 35(6-7), 711-719.
Renault, V., Thornell, L. E., Eriksson, P. O., Butler-Browne, G., & Mouly, V. (2002).
Regenerative potential of human skeletal muscle during aging. Aging Cell, 1(2), 132-139. Taaffe, D. R., Harris, T. B., Ferrucci, L., Rowe, J., & Seeman, T. E. (2000). Cross-sectional and
prospective relationships of interleukin-6 and C-reactive protein with physical
performance in elderly persons: MacArthur studies of successful aging. J Gerontol A Biol
22
Valentine, R. J., Vieira, V. J., Woods, J. A., & Evans, E. M. (2009). Stronger relationship between central adiposity and C-reactive protein in older women than men. Menopause,
16(1), 84-89.
Wei, J., Xu, H., Davies, J. L., & Hemmings, G. P. (1992). Increase of plasma IL-6 concentration with age in healthy subjects. Life Sci, 51(25), 1953-1956.
Vieira, V. J., Hu, L., Valentine, R. J., McAuley, E., Evans, E. M., Baynard, T., & Woods, J. A. (2009). Reduction in trunk fat predicts cardiovascular exercise training-related reductions in C-reactive protein. Brain Behav Immun, 23(4), 485-491.
Vincent, K. R., Braith, R. W., Feldman, R. A., Magyari, P. M., Cutler, R. B., Persin, S. A., Lowenthal, D. T. (2002). Resistance exercise and physical performance in adults aged 60 to 83. J Am Geriatr Soc, 50(6), 1100-1107.
Visser, M., Pahor, M., Taaffe, D.R., Goodpaster, B.H., Simonsick, E.M., Newman, A.B., Nevitt, M., Harris, T.B. (2002) Relationship of interleukin-6 and tumor necrosis factor-alpha with muscle mass and muscle strength in elderly men and woman: the Health ABC study. J Gerontol A Biol Sci Med Sci, 57, 326-332.
Woods, J. A., Wilund, K. R., Martin, S. A., & Kistler, B. M. (2012). Exercise, inflammation and aging. Aging Dis, 3(1), 130-140.
Zammit, P. S., Partridge, T. A., & Yablonka-Reuveni, Z. (2006). The skeletal muscle satellite cell: the stem cell that came in from the cold. J Histochem Cytochem, 54(11), 1177-1191.
