Electrical stimulation (ES) counteracts muscle decline in seniors Helmut Kern, Laura Barberi, Stefan Loefler, Simona Sbardella, Samantha Burgraff, Hannan Fruhmann, Ugo Carraro, Simone Mosole, Nejc Sarabon, Michael Vogelauer, Winfried Mayer, Matthias Krenn, Jan Cvecka, Vanina Romanello, Laura Pietrangelo, Feliciano Protasi, Marco Sandri, Sandra Zampieri and Antonio Musarò
Journal Name:
Frontiers in Aging Neuroscience
ISSN:
1663-4365
Article type:
Original Research Article
First received on:
22 Apr 2014
Frontiers website link:
www.frontiersin.org
Frontiers in Aging Neuroscience
Original Research Date April 22, 2014
Electrical stimulation (ES) counteracts muscle decline in seniors
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Helmut Kern1,2,#, Laura Barberi3,#, Stefan Löfler2, Simona Sbardella3, Samantha Burggraf2, Hannah Fruhmann2, Ugo Carraro2, Simone Mosole2, Nejc Sarabon4, Michael Vogelauer1, Winfried Mayer5, Matthias Krenn5, Jan Cvecka6, Vanina Romanello7, Laura Pietrangelo8, Feliciano Protasi8, Marco Sandri7, Sandra Zampieri2,9, Antonio Musaro3,10,* 1
Institute of Physical Medicine and Rehabilitation, Wilhelminenspital, Vienna, Austria; 2Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation, Vienna, Austria; 3Institute Pasteur Cenci-Bolognetti, DAHFMO- unit of Histology and Medical Embryology, IIM; Sapienza University of Rome, Italy; 4University of Primorska, Science and Research Centre, Institute for Kinesiology Research, Koper, Slovenia; 5Center of Medical Physics and Biomedical Engineering, Medical University of Vienna, Vienna, Austria; 6Faculty of Physical Education and Sport, Comenius University, 81806 Bratislava, Slovakia; 7Dulbecco Telethon Institute at Venetian Institute of Molecular Medicine, Padua, Italy; 8CeSI - Center for Research on Ageing & DNI - Department of Neuroscience and Imaging, University Gabriele d’Annunzio, Chieti, Italy; 9Laboratory of Translation Myology, Department of Biomedical Sciences, University of Padova, Italy; 10Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy #
Equal contribution
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*Correspondence: Prof. Antonio Musarò; Unit of Histology and Medical Embryology, Via A. Scarpa 14 Rome 00161, Italy.
[email protected]
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Keywords: Electrical Stimulation, aging, muscle performance, muscle atrophy, IGF-1, MuRF1, extracellular matrix, satellite cells, microRNA.
Word Count: Abstract = 182, Text = 3.968; Number of references = 70; 2 Tables; 4Figures.
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Abstract
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The loss in muscle mass coupled with a decrease in specific force and shift in fiber composition are
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all marks of aging. Training and regular exercise attenuate the signs of sarcopenia. However,
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pathologic conditions limit the ability to perform physical exercise.
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We addressed whether electrical stimulation (ES) is an alternative intervention to improve muscle
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recovery and defined the molecular mechanism associated with improvement in muscle structure and
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function.
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We analyzed, at functional, structural, and molecular level, the effects of ES training on healthy
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seniors with normal life style, without routine sport activity.
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ES was able to improve muscle torque and functional performances of seniors and increased the size
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of fast muscle fibers. At molecular level, ES induced up-regulation of IGF-1 and modulation of
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MuRF1, a muscle-specific atrophy-related gene. ES also induced up-regulation of relevant markers
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of differentiating satellite cells and of extracellular matrix remodeling, which might guarantee shape
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and mechanical forces of trained skeletal muscle as well as maintenance of satellite cell function,
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reducing fibrosis.
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Our data provide evidence that ES is a safe method to counteract muscle decline associated with
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aging.
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1.
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There is considerable clinical interest in therapeutic strategies to counteract muscle wasting
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associated with aging.
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Skeletal muscle is particularly susceptible to the effects of aging, undergoing a steady reduction in
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function and losing up to a third of its mass and strength. This decline in functional performance is
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due to an overall decrease in muscle integrity, as fibrosis and fat accumulation replace functional
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contractile tissue, and to loss of the fastest most powerful fibers (Scicchitano et al, 2009; Vinciguerra
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et al, 2010).
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At present, it is clear that the most efficient method that has been used to counteract age related
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muscle weakness is long term physical exercise (Paffenbarger et al, 1994). Physical exercise
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increases protein synthesis, turnover and satellite cell number, stimulates appetite, increases IGF-1
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expression levels and capillary bed density. We recently reported that physical exercise in seniors
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preserves muscle morphology and ultrastructure, guarantees a greater maximal isometric force and
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function, and modulates the expression of genes related to autophagy and reactive oxygen species
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detoxification (Zampieri et al, 2014, Mosole S 2014). Nevertheless, certain pathologic conditions and
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aging limit the effectiveness of exercise and therefore the benefits from it.
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An alternative effective intervention to improve muscle recovery is electrical stimulation (ES) (Bax
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et al, 2005, Nuhr et al 2004, Quittan et al 2001, Strasser et al. 2009.). ES has been used in clinical
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settings for rehabilitation purposes, as an alternative therapeutic approach to counteract
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neuromuscular disability, as well as for muscle strengthening and maintenance of muscle mass in
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seniors (Maddocks et al, 2013). In addition, there are studies showing that patients with knee
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osteoarthritis can benefit from the use of ES alone or as an adjunct therapy (Levine et al, 2013;
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Rosemffet et al, 2004). ES directly stimulates skeletal muscle protein synthesis rates (Wall et al,
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2012). Although controversial results have been published as consequence of varying protocols (e.g.
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training 3-7 times a week, training period from 3–12 weeks) and stimulation parameters (e.g.
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stimulation duration 2-30s, stimulation frequency 8-80 Hz) (de Oliveira Melo et al, 2013; Giggins et
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al, 2012), ES represents a promising adjuvant treatment to attenuate muscle disability. Nevertheless
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the molecular mechanisms by which ES exerts its specific anabolic effects on skeletal muscle
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remains to be elucidated.
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Based on our documented clinical experience on the use of ES to rescue permanently denervated
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skeletal muscles in paraplegics (Kern et al, 2004; Boncompagni et al, 2007; Ashley et al, 2007; Kern
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et al, 2008; Kern et al, 2010), we verified whether ES can be proposed as a therapeutic tool to
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rehabilitate skeletal muscle of sedentary seniors.
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We demonstrated that ES mimics the beneficial effects of physical exercise in muscle of ageing
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individuals and we defined the molecular signature underlying these effects.
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2.
Materials and methods
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2.1. Subjects enrolled in the study
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Sixteen subjects (8 male and 8 female) (73.1 ± 6.9 [y], 81.7 ± 14.7 [kg], 170.3 ± 11.2 [cm]) were
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recruited for the study. All of the subjects were volunteers who signed an informed consent and
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received detailed information about the functional test protocols, the trainings and muscle biopsies.
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Approval from the national committee for medical ethics was obtained at the beginning of the study
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(EK08-102-0608). All subjects included were healthy and declared not to have any specific
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physical/disease issue.
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2.2.Electrical stimulation training
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Subjects were exposed to regular neuromuscular ES training (swelling current) for a period of 9
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weeks, starting two times a week for the first 3 weeks and then switched to 3 three times a week for
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the next 6 weeks, amounting to a total of 24 training sessions (3x10 minutes each session). Electrical
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stimulation training was performed with a two channel custom-built battery-powered stimulator
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(Krenn et al, 2011) at home by the subjects themselves after detailed instructions. The subjects
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applied two conductive rubber electrodes (9 × 14 cm; 126cm²) which were attached to the skin by
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wet sponge on the anterior thigh on both sides (left/right). The electrode pairs for left and right thigh
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were connected to the two channels of the stimulator. This allowed independent activation of the left
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and right thigh muscles, which were stimulated in an alternative manner. Each repetition (i.e. ES
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evoked muscle contraction) was evoked by a 3.5 s train (60 Hz) of electrical pulses (rectangular,
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biphasic, width 0.6 ms). Consecutive contractions of the same thigh were separated by 4.5 s off
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intervals. In this study constant voltage stimulation devices were applied. The subjects were
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instructed to increase the stimulation intensity until their maximum sensory tolerance level was
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reached. With this intensity all of the subjects achieved full knee extension. Nevertheless, the applied
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current and voltage was recorded by the stimulation device for each training session. The mean
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stimulation current was 128 +/- 16mA and voltage of 39 +/- 14V.
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2.3. Force measurement
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An isometric measurement on a dynamometer (S2P ltd., Lubljana, Slovenia) as described (Šarabon et
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al, 2013a; Šarabon et al, 2013b) with 90° hip flexion and 60° knee flexion (full knee extension = 0°)
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was performed 3 times at each leg to assess the maximal isometric torque of the left and right knee
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extensors. The best value of each leg was taken for further analyses.
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2.4. Functional Tests
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A complete set of functional tests to access mobility and function in activities of daily living (ADL)
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was designed and applied to each of the subjects. These tests included: time up and go test (TUGT)
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(Podsiadlo and Richardson, 1991) where the subjects were asked to stand up from a standard chair,
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walk a distance of 3 meters, turn around, walk back to the chair and sit down again all as fast as
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possible; short physical performance battery (SPPB) (Guralnik et al, 1994) to evaluate the lower
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extremities function by using tests of gait speed (2,4 m), standing balance (side-by-side, semi-tandem
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and tandem stance for 10 s) and the time which the subject needed to rise from a chair for five times
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as quickly as possible with the arms folded across their chest; 12 flight Stair Test (Suzuki et al, 2001)
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where the participant was instructed to ascend and descend the stairs after reaching the top (12th)
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step as quick and safe as possible; and 10m-walking test with habitual and fastest walking speed (but
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not running) (Šarabon et al, 2010a; Šarabon et al, 2010b) where each speed was performed three
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times, the time was measured and average velocity calculated.
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2.5. Muscle biopsies
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Muscle biopsies were harvested as described (Kern et al, 2004) from the vastus lateralis muscle 15-
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20 cm proximal of the joint space of the knee, with the Bergström needle inserted perpendicular to
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the fibre direction. The biopsies before training were taken 10 days after the initial assessment at
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inclusion to the study, electrical stimulation training started 14 days later. Post training biopsies were
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taken 7 days after the last training session. The final functional assessment was done 4 days after the
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last training session. About 50-70mg of tissue was harvested from both legs of the subjects.
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2.6.1. Histological analysis
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Serial cryosections (8 microns thickened) from frozen muscle biopsies were mounted on polysine™
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glass slides, air-dried and used for further analyses. For morphometric analysis, cryosections were
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stained with hematoxylin-eosin (HE),whereas ATPases protocol at pH 4.35 was used to visualize
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slow-type (dark-stained) and fast-type muscle fibers (light-stained).
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2.6.2. Morphometric analysis
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The mean myofiber diameter and the percentage of slow and fast type muscle fibers were evaluated
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from stained cross sections in accordance with our previous published methods (Rossini K et al.
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2002, Kern et al, 2008; Ashley et al, 2007; Kern et al, 2010; Carraro et al, 2005; Biral et al, 2008).
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Images were acquired using a Zeiss microscope connected to a Leica DC300F camera. Morphometry
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analysis was performed using Scion Image software (2000 Scion Corporation, Inc).
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2.7. Immunofluorescence analysis
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Muscle sections were incubated either for 1 hour at room temperature (RT) or overnight at 4°C, with
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anti-neural adhesion molecule (N-CAM) rabbit polyclonal antibody (Chemicon, Italy), anti-Pax7
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mouse monoclonal antibody (DSHB, Iowa), or anti-laminin rabbit polyclonal antibody (Sigma, Italy)
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1:100 diluted in PBS, respectively, as described (Zampieri et al 2010, Mosole et al 2014). Sections
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were then incubated for 1 hour at RT with Cy3 or Alexa Fluor® 488 dye conjugated antibodies
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against rabbit (Chemicon, Italy) or mouse IgG (Life technologies, Italy). Sections were then
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mounted on glass slides using ProLong Gold antifade reagent with DAPI (Life Technologies).
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Quantitation of Pax7 positive cells were performed on captured images from random fields counting
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a minimum of 300 fibers per biopsy.
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2.8. Electron Microscopy (EM) and quantitative analyses of calcium release units (CRUs) and
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mitochondria
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Biopsy specimens were fixed, sectioned and imaged by EM as previously described (Boncompagni
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et al, 2006; Boncompagni et al, 2007). Number/area of CRUs and mitochondria was determined
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from electron micrographs of non-overlapping regions randomly collected from longitudinal
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sections as in (Boncompagni et al, 2007).
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2.9. Gene expression analyses and miRNA
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Total RNA extraction from human muscle biopsies before and after ES was performed with tissue
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lyser (Qiagen) in TriRiagentTM (Sigma) and small RNAs were purified using PureLink miRNA
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Isolation Kit (Invitrogen). This RNA fraction, containing miRNA, was reverse-transcribed using the
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TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies); the other RNA fraction,
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containing mRNA, was reverse-transcribed using a QuantiTect Reverse Transcription kit (Qiagen).
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The reverse-transciption reactions were performed according to the manufacturers’ instructions.
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Quantitative PCR was performed on an ABI PRISM 7500 SDS (Applied Biosystems, USA), using
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premade 6-carboxyfluorescein (FAM)-labeled TaqMan assays for GAPDH, IGF-1 Ea, IGF-1 Eb,
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IGF-1 Ec, IGF-1 pan, Myostatin, Collagen I, III, VI (Applied Biosystems, USA). FAM-labeled
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TaqMan MicroRNA Assays for miR-1, miR-133a, miR-206, miR-29, and U6 snRNA (Applied
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Biosystems, USA) were performed as described. Quantitative RT-PCR sample values were
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normalized to the expression of GAPDH mRNA or U6 snRNA. The relative level for each gene and
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miRNA was calculated using the 2-DDCt method (Livak and Schmittgen, 2001) and reported as
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mean fold change in gene expression.
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2.10. Statistical Analyses
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SPSS Statistics software package, version 17.0 was used to evaluate differences between the
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measurements in parameters of torque, functional tests, muscle morphometry and molecular data.
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Normal distribution was obtained with Shapiro-Wilk-Test, the two-tailed paired and unpaired
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Student’s t test and Wilcoxon-Test were used for normal and not normal distributed variables,
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respectively. The level of significance was set to p
