Ultrasound in Med. & Biol., Vol. 40, No. 3, pp. 504–512, 2014 Copyright Ó 2014 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter
http://dx.doi.org/10.1016/j.ultrasmedbio.2013.10.013
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Original Contribution INDUCTION OF SKELETAL MUSCLE DIFFERENTIATION IN VITRO BY THERAPEUTIC ULTRASOUND VIVIANE MENDES ABRUNHOSA,*y CAROLINA PONTES SOARES,y ANA CLAUDIA BATISTA POSSIDONIO,y ANDRE VICTOR ALVARENGA,* RODRIGO P. B. COSTA-FELIX,* MANOEL LUIS COSTA,y and CLAUDIA MERMELSTEINy * Laboratorio de Ultrassom, Diretoria de Metrologia Cientıfica e Industrial (DIMCI), Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMETRO), Rio de Janeiro, RJ, Brazil; and y Instituto de Ci^encias Biomedicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil (Received 17 July 2013; revised 11 October 2013; in final form 15 October 2013)
Abstract—Therapeutic ultrasound (TU) has been used for the last 50 y in rehabilitation, including treatment of soft tissues. Ultrasound waves can be employed in two different modes of operation, continuous and pulsed, which produce both thermal and non-thermal effects. Despite the large-scale use of TU, there are few scientific studies on its biologic effects during skeletal muscle differentiation. To better analyze the cellular effects of TU, we decided to follow cells in vitro. The main purpose of this study was to evaluate the effects of TU in primary chick myogenic cell cultures using phase contrast optical microscopy and immunofluorescence microscopy, followed by image analysis and quantification. Our results indicate that TU can stimulate the differentiation of skeletal muscle cells in vitro, as measured by the thickness of multinucleated myotubes, the ratio of mononucleated cells to multinucleated cells and expression of the muscle-specific protein desmin. This study is a first step toward a metrologic and science-based protocol for cell treatment under different ultrasound field exposures. (E-mail: [email protected]) Ó 2014 World Federation for Ultrasound in Medicine & Biology. Key Words: Therapeutic ultrasound, Skeletal muscle differentiation, Desmin, Myogenesis.
or non-thermal (pulsed mode) (Dyson 1987). Thermal effects in vivo are derived from the conversion of ultrasonic energy into elevation of the tissue temperature between 40 C and 45 C for at least 5 min. These effects have been related to increased blood flow and flexibility of collagen fibers, reduction in muscle spasm and a proinflammatory response (Speed 2001). Non-thermal effects are the local vibrations that involve stable acoustic streaming and cavitations in tissue fluids and could improve tissue healing (Chan et al. 2010). Both effects, thermal and non-thermal, occur simultaneously during TU treatment. The administration of TU involves the choice of frequency, mode of operation of the equipment, effective intensity levels and duration of treatment. TU is broadly used in the frequency range of 0.5 to 5.0 MHz, but typically TU equipment can be set only at 1 or 3 MHz. Different frequencies are recommended depending on the injury and subcutaneous fat tissue of the patient. Frequencies of 1 MHz are absorbed primarily by tissues at a depth of 3–5 cm, and frequencies of 3 MHz are absorbed at a depth of 1–2 cm. The choice of mode of operation of
INTRODUCTION Soft tissue lesions are common in athletic and nonathletic populations and can be caused by direct contusions, indirect strain injuries or intensive exercise. These injuries affect muscle function, joint mobility and, thereby, quality of life (Rantanen et al. 1999). One of the most recommended treatments for a wide variety of skeletal muscle tissue injuries is therapeutic ultrasound (TU) (Speed 2001). Therapeutic applications of ultrasound have been widely used in physiotherapy since the 1950s (Shaw and Hodnett 2008; Watson 2008). Ultrasound is a form of mechanical energy with a highfrequency pressure wave that can be transmitted into the body (Khanna et al. 2008). The interaction of the ultrasound wave with the tissue can promote biophysical effects that can be classified as thermal (continuous mode)
Address correspondence to: Claudia Mermelstein, Universidade Federal do Rio de Janeiro, Instituto de Ci^encias Biomedicas, Av. Carlos Chagas Filho 373, Ilha do Fund~ao, 21941-902 Rio de Janeiro, RJ, Brazil. E-mail: [email protected] 504
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the equipment depends on the goal of the treatment. Continuous mode could cause a larger temperature increase in the tissue than pulsed mode, with other parameters being held constant. Although the mode of operation is related to temperature in the tissue, the intensity level is also involved. The effective intensity (W/cm2) is acquired from the quotient of maximum ultrasonic output power, delivered through the patient’s body, and the transducer’s effective radiating area. Dyson (1987) found that elevation of temperature in the tissue occurs in both continuous and pulsed mode. The other parameter involved in TU is the duration of treatment. There is no consensus on the duration of TU treatment, which can vary between 2 and 15 min for treatment of disorders in soft tissues. The amount of energy delivered to the tissue is proportional to the duration of treatment (Robertson and Baker 2001). The studies that have examined the effects of TU in soft tissues reported divergent results with respect to the efficacy of TU treatment. The variability in the efficacy of TU treatment could be caused by the lack of standardization in TU protocols and/or by the fact that different biologic models were used (Fisher et al. 2003; McBrier et al. 2007; Silveira et al. 2012). Although some of these studies were conducted in animal models, most were performed using the C2C12 mouse myoblast cell line. To better analyze the effects of TU at the cellular level, we studied primary skeletal muscle cell culture. Primary cultures of skeletal muscle cells are more similar to the in vivo system than is any muscle cell lineage. Thus, our main goal was to investigate the relationship between the protocols commonly used for TU treatment for muscle lesion recovery and their effects on skeletal muscle differentiation. The experiments were performed in primary cultures of chick skeletal muscle cells using TU equipment widely employed in physiotherapy. The chick myoblast primary culture is a robust in vitro model of myogenesis. Skeletal myogenesis proceeds in these cell cultures through the following main sequential stages: myoblast proliferation, cell cycle withdrawal, myoblast alignment, and fusion and their subsequent differentiation into striated multinucleated myotubes. The fact that these steps have been well characterized makes this an interesting model for analysis of the effects of TU during muscle differentiation. Regeneration of an injured skeletal muscle involves the activation of the proliferation of satellite cells, followed by their differentiation and fusion into myotubes, which contributes to the functional recovery of the muscle tissue. Differentiation of satellite cells recapitulates the embryonic myogenesis. The molecular and cellular basis of satellite cell differentiation can therefore be studied in embryonic myoblast cell culture models. One of the aims of our study was to determine whether TU treat-
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ment can activate the proliferation and differentiation of embryonic myoblasts, assuming that this process would be equivalent in muscle injury-activated satellite cells, and thus contribute to the comprehension of TU effects on muscle lesion healing. Our results indicated that skeletal muscle cell differentiation is induced in vitro by therapeutic ultrasound. METHODS Antibodies and fluorescent probes The DNA-binding probe 4,6-diamidino-2phenylindole dihydrochloride (DAPI), Alexa Fluor 488conjugated goat anti-mouse serum and Alexa Fluor 546-conjugated goat anti-rabbit serum were purchased from Molecular Probes (Eugene, OR, USA). Rabbit polyclonal anti-desmin (D-8281), mouse monoclonal antisarcomeric a-actinin (clone EA-53, A-7811) and mouse monoclonal anti-a-tubulin (clone DM1A, T-9026) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrasonic pressure field mapping system A water bath measuring 1700 3 1000 3 800 mm from Ultrasound Labs (National Institute of Metrology, Quality and Technology, Inmetro, Rio de Janeiro, Brazil) was used for mapping the ultrasound pressure field of the transducer. The specified positioning system (Newport, Irvine, CA, USA) used to move the transducer (or hydrophone) in the water bath allows movements of 300 mm along the x- and y-axes and 600 mm along the z-axis. The transducer is excited using a 20-cycle burst of sine wave generated by a function generator (AFG 3252, Tektronix, Beaverton, OR, USA), and waterborne signals are acquired using an oscilloscope (TDS 3032B, Tektronix). In the mapping procedure, a needle hydrophone is used with an active element of 0.2 mm (Precision Acoustics, Dorchester, Dorset, UK). To integrate all system components, and also to provide a user-friendly interface, virtual software was developed in the LabView platform (National Instruments, Austin, TX, USA). The software automatically performs the raster scan necessary to calculate the effective radiating area (AER) of the physiotherapeutic ultrasound transducer (Alvarenga and Costa-Felix 2009). Primary myogenic cell culture This study using chick embryos was approved by the Ethics Committee for Animal Care and Use in Scientific Research of the Federal University of Rio de Janeiro and received Approval No. DAHEICB004. All cell culture reagents were purchased from Invitrogen (S~ao Paulo, Brazil). Primary cultures of myogenic cells were prepared from breast muscles of 11-d-old chick embryos (Mermelstein et al. 2005). Cells were plated at an initial density of 106 cells/35 mm in culture dishes coated with
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rat tail collagen. Cells were grown with 2 mL of culture medium (Minimum Essential Medium supplemented with 10% horse serum, 0.5% chick embryo extract, 1% L-glutamine, and 1% penicillin-streptomycin) under a humidified 5% CO2 atmosphere at 37 C. Twenty-fourhour cultures were treated with therapeutic ultrasound waves (see Table 1). Sodium bicarbonate (1.4 g/L) was added to the culture medium to maintain pH in the range of 7.2–7.4. Phenol red, a pH indicator, was added to medium to colorimetrically monitor changes in pH in both untreated and ultrasound-treated cells. After treatment, cultures were grown for the next 48 h and culture medium was changed every day. Treatment with ultrasound wave An ultrasound device (Model Sonopulse Compact, Ibramed, Amparo, Brazil) was used in different modes of operation (continuous or pulsed) at different effective intensities (0.5 or 1 W/cm2) for different durations of treatment (5 or 10 min). The frequency of the TU was the same for all treatments (1 MHz). Ultrasound was transmitted through the bottom of the culture dish, with a coupling gel between the ultrasound transducer and the dish. The ultrasonic attenuation effects within the medium were disregarded, as they were the same throughout all cell cultures and different treatments, and the free path is markedly short (about 2 mm). Most likely, reflections on the free surface of the medium will result in an identical ultrasonic field inside the medium, as all culture dishes were prepared in a similar way with respect to the amount (volume) of medium. Both attenuation and surface reflections were considered inherent characteristics of the ultrasonic pressure field and very similar to each other in all tested cultures. The diameter of the ultrasound transducer was 35 mm (the same as the culture dish). All 24-h cell cultures were exposed to TU treatment only once. Control cultures were subjected to the same experimental procedures as the treated cultures, except that the ultrasound Table 1. Protocols for ultrasound wave treatment* Mode of operation Continuous Continuous Continuous Continuous Pulsed Pulsed Pulsed Pulsed
Intensity (W/cm2)
Duration of treatment (min)
0.5 0.5 1.0 1.0 0.5 0.5 1.0 1.0
5 10 5 10 5 10 5 10
* An ultrasound apparatus was used in different modes of operation (continuous or pulsed) at different effective intensities (0.5 or 1 W/ cm2) for different durations of treatment (5 or 10 min). The frequency of therapeutic ultrasound was the same for all treatments (1 MHz).
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equipment was not turned on (sham ultrasound). The different TU treatments used in cells are described in Table 1. All analyses were performed 48 h after treatment (which means that the cells were grown in culture for a total of 72 h). The ultrasound apparatus was certificated according to IEC 61689:2007 before its use in this study. The temperature of the culture medium was measured immediately before and after TU treatment. Temperature was measured using an infrared thermometer (Model 62MAX1, Fluke, Beijing, China) directly on the medium of each dish without the cover lid. Cell viability Cell viability was measured with the XTT assay (Scudiero et al. 1988). XTT (2,3-bis (2-methoxy-4-nitro5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide, Sigma-Aldrich, USA) is metabolically reduced in viable cells to a water-soluble formazan product. This reagent allows direct absorbance readings. XTT was prepared at 1 mg/mL in pre-warmed (37 C) Minimum Essential Medium without serum. PMS (phenazine methosulfate, Sigma-Aldrich) was prepared at 5 mM (1.53 mg/mL) in phosphate-buffered solution (PBS). Fresh XTT and PMS were mixed together in the appropriate concentrations. For a 0.025 mM PMS-XTT solution, 25 mL of the stock 5 mM PMS was added per 5 mL of XTT (1 mg/mL). One hundred microliters of this mixture (final concentration, 50 mg XTT and 0.38 mg PMS per well) was added to each of the 96 wells on the plate containing 24-h myogenic cell cultures (untreated and TU-treated). After incubation at 37 C for 24 h, the absorbance at 492 nm was measured with a SunriseBasic spectrophotometer (Tecan, Gr€odig, Austria). Immunofluorescence microscopy and digital image acquisition Myogenic cells were rinsed with PBS for 10 min at room temperature. Cells were then permeabilized with 0.5% Triton-X 100 in PBS three times for 10 min. The same solution was used for all subsequent washing steps. Cells were incubated with primary antibodies for 1 h at 37 C in a humidity chamber. After incubation, cells were washed for 30 min and incubated with Alexa Fluor 488 and/or Alexa Fluor 546-conjugated secondary antibodies for 1 h at 37 C in a humidity chamber. After a 10-min wash with 0.9% NaCl, DAPI (0.1 mg/mL in 0.9% NaCl) was added for 5 min. Cells were mounted in ProLong Gold anti-fade reagent (Molecular Probes) and examined with an Axiovert 100 microscope (Carl Zeiss, Jena, Germany) by using filter sets that were selective for each fluorochrome wavelength channel. Images were acquired with a DP72 camera (Olympus, Tokyo, Japan). All nuclei labeled with DAPI were counted in mononucleated and multinucleated desmin-positive cells.
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Quantification of cultured images Phase contrast microscopy images of live cultured cells were acquired with an Axiovert 100 microscope (Carl Zeiss). Images of 72-h myogenic cultures were acquired from several equidistant microscopic fields, representative of the whole dish for each culture condition. Diameter of myotubes and number of nuclei were obtained using the public domain software ImageJ (Schneider et al. 2012). Myotube thickness was measured in the thicker region of each myotube, for all myotubes in each microscopic field, in at least 50 different fields for each experimental condition. The data were collected from three independent experiments.
Statistical analysis Data are presented as the mean 6 standard deviation. Mean values were calculated from at least three independent experiments. Tests of statistical significance were performed using IBM SPSS software. Statistical analysis of the data related to myotube thickness was performed by one-way analysis of variance. An unpaired t-test was used for the data related to the nucleus quantification. Statistical comparisons of the immunoblot data were performed with Student’s t-test. The values were considered to be statistically different at p-values # 0.05.
SDS-PAGE and immunoblotting Untreated and TU-treated myogenic cultures were grown for 72 h. Cultures were then quickly washed in ice-cold PBS. Fifty microliters of ice-cold sample buffer (4% sodium dodecyl sulfate [SDS], 20% glycerol, 0.2 M dithiothreitol, 125 mM Tris-HCl, pH 6.8) was added to the cultures, and then cells were scraped off the dish with a plastic cell scraper. Cell extracts were recovered in a tube, boiled for 5 min at 95 C and centrifuged at 12,000g for 15 min. The pellet was discarded and the supernatant was used in subsequent steps. The amount of protein in each sample was determined according to the Bradford (1976) method, using bovine serum albumin as a standard. Equal amounts of protein were loaded on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to poly (vinyl difluoride) membranes. The proteins immobilized on the membranes were immediately blocked for 1 h at room temperature with 5% non-fat dry milk in Tris-buffered saline-Tween 20 solution (0.001%) (TBS-T). Then the membranes were incubated for 12 h at 37 C with an anti-desmin polyclonal antibody. After five washes in TBS-T (3 min each), the membranes were incubated for 1 h at 37 C with an anti-rabbit peroxidase-conjugated antibody (dilution 1:10,000 in TBS-T; Amersham, GE Healthcare, Pittsburgh, PA, USA) and washed again as described above; the bands were visualized using the ECL Plus Western Blotting Detection System (Amersham/GE Healthcare). To check sample loading, other membranes with the same samples were incubated with a mouse monoclonal anti-a-tubulin antibody (Sigma-Aldrich, dilution 1:3000 in TBS-T-milk). After five washes in TBS-T (3 min each), membranes were incubated with anti-mouse peroxidase-conjugated antibody (Amersham, dilution 1:10,000 in TBS-T) and developed as described above. Quantification of protein bands was performed using the public domain software ImageJ (Schneider et al. 2012) with data obtained from three independent experiments.
Therapeutic ultrasound transducer reveals a non-homogeneous pressure field First, we established the effectiveness of the ultrasound system used by evaluating the ultrasound pressure field of the transducer by mapping the ultrasound field in an acoustic tank. The transducer is non-focused; that is, it has a natural focus. Its last maximum of ultrasonic pressure amplitude was measured at 109.5 6 2.7 mm from its surface. The transducer’s mapping disclosed non-homogeneity over its output surface, but the nonuniformity ratio of the ultrasonic beam was less than 8. It leads to non-uniform output, but without marked hot spots. Moreover, the medium depth is short compared with the transducer diameter (2 and 35 mm, respectively), which results in near-field behavior throughout the medium. In this case, diffraction interference is not expected to prevail, despite the multiple reflections and attenuation that might occur. The average value of AER for the physiotherapy transducer used in this study was 2.43 cm2, with an estimated expanded uncertainty of 2.9 3 10 cm2
RESULTS
Fig. 1. Cell viability of untreated and therapeutic ultrasound (TU)-treated myogenic cell cultures. Myogenic cells were grown for 24 h, subjected to sham ultrasound (control) or different TU treatments and grown for the next 24 h. Cell viability was quantified by the XTT assay (Scudiero et al. 1988) assay in three independent experiments. Note that cell viability did not change significantly after ultrasound treatment. Treatment parameters are given as continuous (cont) or pulsed (pul) mode–intensity (W/cm2)–duration (min).
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Fig. 3. Mean thickness of myotubes in untreated and therapeutic ultrasound (TU)-treated myogenic cell cultures. Myogenic cells were grown for 24 h, subjected to sham ultrasound (control) or different TU treatments and grown for the next 48 h. Myotube thickness was measured in at least 50 different microscopic fields for each experimental condition from three independent experiments. Note that some TU treatments induced significant enhancement in the thickness of myotubes. *p , 0.05; one-way analysis of variance, compared with control, n 5 3. Treatment parameters are given as continuous (cont) or pulsed (pul) mode–intensity (W/cm2)–duration (min).
guideline, this study was framed in level 1, because the results were referred to the physiotherapy system’s nominal ultrasonic intensity instead of the actual acoustic output.
Fig. 2. Phase contrast microscopy analysis of untreated and therapeutic ultrasound (TU)-treated myogenic cell cultures. Myogenic cells were grown for 24 h, subjected to sham ultrasound (control) or different TU treatments and grown for the next 48 h. Experimental conditions are detailed in Table 1. One myotube in each image is in green. The yellow line in the image of cells treated with continuous mode ultrasound at 0.5 W/cm2 for 5 min indicates how myotube thickness was measured. Treatment parameters are given as continuous (cont) or pulsed (pul) mode– intensity (W/cm2)–duration (min). Bar 5 50 mm.
(11.9%). To understand the relationship between exposure conditions and any observed biologic effects, a guideline was proposed by ter Haar et al. (2011). According to this
TU does not affect cell viability in myogenic cultures Next, we tested the effects of the continuous and pulsed modes of operation, two different intensities (0.5 and 1.0 W/cm2) and two different durations of treatment (5 and 10 min) in a primary culture of chick embryonic skeletal muscle cells. The treatments tested here were chosen by taking into consideration the protocols most commonly used in clinical practice. To determine whether TU treatment has any effect on cell viability, experiments were performed with untreated and TU-treated myogenic cultures grown 24 h after TU exposure, using a XTT-based method (Scudiero et al. 1988). No significant effect on cell viability was noted in the myogenic cells 24 h after TU treatment compared with untreated cells (Fig. 1). We also measured the temperature of the culture medium after TU treatment and did not observe an increase in temperature. TU induces differentiation of embryonic skeletal muscle cells Phase contrast microscopy revealed that some TU treatments enhanced muscle differentiation, as suggested by an increase in myotube thickness (Fig. 2). TU
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Fig. 4. a-Actinin distribution in untreated and therapeutic ultrasound (TU)-treated myogenic cell cultures. Myogenic cells were grown for 24 h, subjected to sham ultrasound (a, c) or continuous ultrasound mode at 0.5 W/cm2 for 5 min (cont–0.5–5) and grown for the next 48 h (b, d). Cells were fixed and double-stained with an anti-sarcomeric a-actinin antibody (green, a–d), and the nuclear dye 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue, a and b). A merged image is provided in (a) and (b). Note that cultures treated with cont–0.5–5 (d) had thicker myotubes than untreated cells (c). a-Actinin (in green) in localized in the Z-bands of striated myofibrils in both untreated and ultrasound-treated myotubes. Myotube thickness is represented by white line in (d). Bar 5 100 mm (a, b) and 20 mm (c, d).
treatment in continuous mode at 0.5 W/cm2 for 5 or 10 min (cont–0.5–5 and cont–0.5–10), TU in continuous mode at 1.0 W/cm2 for 5 min (cont–1.0–5) and TU in pulsed mode at 1.0 W/cm2 for 5 min (pul–1.0–5) differed statistically significantly (95% confidence interval), as measured by myotube thickness, compared with no treatment (Fig. 3). To confirm whether or not the cont–0.5–5 treatment enhanced muscle differentiation, we analyzed the expression and distribution of the muscle-specific marker a-actinin. We decided to test only the cont–0.5–5 treatment because our previous results (Figs. 2 and 3) had indicated that this TU treatment promoted major changes in myotube thickness. a-Actinin is a myofibrillar protein that localizes in Z-bands in sarcomeres. Immunofluorescence images revealed the presence of a-actinin in striated myofibrils in multinucleated myotubes in both untreated and ultrasound-treated cultures (Fig. 4). Cell cultures subjected to cont–0.5–5 displayed more and thicker myotubes, confirming the enhancement in skeletal muscle differentiation. It is possible to observe a larger number of nuclei (labeled with the nuclear dye DAPI) in the TU-treated condition (Fig. 4a, b), which could be inter-
preted as an increase in cell proliferation after TU exposure. These results indicated once again that cont–0.5–5 induced the formation of thicker myotubes compared with no treatment. We also quantified the number of nuclei in mononucleated and multinucleated (myotubes) cells in untreated cultures and cultures treated with cont–0.5–5. Our results indicate that cont–0.5–5 induced a 30% increase in the total number of nuclei per field and a 60% increase in the total number of nuclei per myotube (Fig. 5). Note also that the proportion of nuclei in mononucleated cells, compared with nuclei in myotubes, is even higher in TU-treated cultures (from 1.5 to 3.1). The increase in the total number of cells in TU-treated cultures could indicate enhancement of cell proliferation and/or survival. The increase in the ratio of mononucleated cells to multinucleated cells suggests enhancement of cell fusion. Next we decided to quantify the amount of desmin protein expression in untreated cells and cells treated with cont–0.5–5. Cells were grown for 24 h, treated with TU and grown for the next 48 h. Untreated and TUtreated cell extracts were analyzed by 10% SDS-PAGE
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Fig. 5. Average number of nuclei in untreated and therapeutic ultrasound (TU)-treated myogenic cell cultures. Myogenic cells were grown for 24 h, subjected to sham ultrasound (control) or continuous ultrasound mode at 0.5 W/cm2 for 5 min (cont–0.5–5) and grown for the next 48 h. The nuclei present in mononucleated cells and multinucleated cells (myotubes) were quantified in at least 50 different microscopic fields for each experimental condition from three independent experiments. Note that cont–0.5–5 induced a 30% increase in the total number of nuclei per field and a 60% increase in the total number of nuclei per myotube. *p , 0.05, unpaired t-test, compared with control, n 5 3.
followed by immunoblotting. Quantification of immunoblots revealed a 60% increase in the expression of desmin (52 kDa) in TU-treated cells compared with control cells (Fig. 6). DISCUSSION In the work described here, we studied the effects of commonly used TU treatment protocols for muscle lesion recovery in chick skeletal muscle cell culture to determine the relationship between TU protocols and their effects on muscle differentiation. We found that several of these protocols, most of them in continuous mode, enhance skeletal muscle fiber formation. Muscle differentiation was reflected in the presence of more myotubes with more well-organized striated myofibrils. The extent of differentiation was analyzed by measurement of the thickness of multinucleated myotubes and the ratio of mononucleated cells to multinucleated cells. These findings argue that the chick in vitro model of myoblast cell culture is a valuable tool for studies of the cellular and molecular effects of TU during muscle differentiation. In vitro models such as primary chick myogenic culture begin with a population of replicating mononucleated myoblasts and some fibroblastic cells (Holtzer et al. 1991). After a few rounds of replication, these myoblasts withdraw from the cell cycle and begin to change in
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Fig. 6. Therapeutic ultrasound (TU) enhances the expression of desmin in myogenic cell cultures. (a) Total cell extracts from primary cultures of chicken myogenic cells were analyzed by Western blotting using an anti-desmin antibody. Cells were grown for 24 h, subjected to sham ultrasound (control) or continuous ultrasound mode at 0.5 W/cm2 for 5 min (cont–0.5–5) and grown for the next 48 h. (b) Quantification of immunoblots revealed a 60% increase in the levels of desmin expression (52 kDa) in cells after TU treatment compared with control cells. The semi-quantitative analysis represents the average of three independent experiments. *p , 0.05, Student’s t-test, compared with control, n 5 3.
shape, becoming elongated and bipolar cells. Bipolar myoblasts then enter a cell-cell recognition phase in which their plasma membranes adhere and fuse. Fusion gives rise to multinucleated and striated myotubes. The enhancement in thickness of myotubes and number of nuclei per myotube after TU treatment could be caused by an increase in myoblast fusion. Myoblast fusion can be enhanced in different ways, such as an increase in membrane fluidity and/or permeability, an increase in myoblast proliferation (which occurs before fusion) and exposure or removal of membrane molecules that are necessary for the fusion process. It has been proposed that TU treatment induces changes in cell membrane permeability, a property that is widely used for improving gene transfer protocols (Li et al. 2012). Few studies have examined the effect of TU in the treatment of skeletal muscle (Fisher et al. 2003; McBrier et al. 2007; Plentz et al. 2008), and none of them found significant differences using continuous mode treatment. Our results contradict those articles, because we found that all continuous mode TU treatments, with the exception of cont–1.0–10, induced
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enhancement of myotube thickness. Several explanations for these contradictory findings can be hypothesized. One possible explanation might be the difference between their in vivo approach and our in vitro approach. Fewer variables are involved in myogenesis in skeletal muscle cell cultures than in animal myogenesis. In an animal, there are hundreds of cell types interacting with each other and with the extracellular matrix, through the exchange of chemical signals in a 3-D environment. In contrast, cell cultures respond to a reduced range of stimuli in a controlled environment. Therefore, cell culture models are usually less variable and provide higher reproducibility than animal models. In addition, the results obtained in cell cultures are produced directly by the cells and their controlled environment, whereas in animals, the results could arise from complex interactions between tissues and/or systems. Other possible explanations for the contradictory findings between our results and those of other authors are: (i) differences between species (birds vs. mammals); (ii) differences between a muscle damage recovery model and our normal myogenesis model; and (iii) differences in TU conditions used, such as, frequency, intensity of the equipment and duration of treatment, as suggested by Schuster et al. (2013). The enhancement in myogenesis we observed is probably not related to the thermal effect of the continuous or pulsed mode condition used, as no increase in temperature was detected in the myogenic cell cultures after TU treatments. Most of the cell cultures we treated with pulsed TU did not exhibit an increase in myotube thickness, with the exception of pul–1.0–5, whereas myotube thickness increased in most continuous TU cultures. This finding may be explained by the fact that the energy delivered to the culture by pulsed mode at 1.0 W/cm2 is the same as that delivered by continuous mode at 0.5 W/cm2 during the 5 min in which we observed positive effects on muscle thickness. Interestingly, we could not observe any correlation between the overall energy delivered by TU to cells and their differentiation. Our results are in agreement with those of Fisher et al. (2003), who reported a significant increase in synthesis of contractile proteins in injured gastrocnemius of male Sprague-Dawley rats after treatment with the same of mode of operation (pulsed), intensity (1.0 W/cm2) and duration of treatment (5 min) used in our study. Studies using TU conditions similar to those described above have suggested that these results are relative to an increase in satellite cell proliferation (Rantanen et al. 1999). In contrast to our results, Ikeda et al. (2006) reported that low-intensity pulsed ultrasound (LIPUS, 1.5 MHz, 70 mW/cm2 for 20 min) induces a significant decrease in expression of the myogenic transcription factor
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MyoD in the myoblast cell line C2C12. In agreement with our data, Nagata et al. (2013) described that treatment of C2C12 cells with TU (LIPUS, 3 MHz, 30 mW/ cm2 for 15 min) up-regulated expression of the myogenic transcription factor myogenin, and treatment of mouse muscle with TU induced an increase in the crosssectional area of myofibers. Park et al. (2010) reported that C2C12 cells subjected to LIPUS (30 mW/cm2 for 3 or 10 min daily for 6 d) exhibited a significant increase in expression of tropomyosin. Thus, we can conclude from the results presented here with primary cultures of chick myoblasts and from the data previously described by other groups with the C2C12 cell line (Nagata et al. 2013; Park et al. 2010) that some TU treatments can promote myogenesis and, thus, can be a valuable tool in treatment of muscle injuries. It is difficult to draw further conclusions from these studies because of differences in the test systems. Furthermore, inadequate calibration of TU equipment can also interfere with treatment. It has been reported that performance of TU equipment that is more than 10 y old and equipped with dual-frequency treatment heads can be compromised (Speed 2001). To avoid these problems, we sought to certify our TU equipment. We acknowledge the differences between treating muscle cells in vitro with TU and treating human muscle tissues in vivo. In addition to the fact that cell cultures lacks the complexity of muscle tissues, as mentioned above, there is also the problem of artificial interactions between the acoustic field and the plastic culture dish. In the present work, we used a coupling gel between the TU transducer and the plastic culture dish to minimize these unwanted interactions. On the other hand, as we illustrated the direct effects of TU on cell differentiation, cell cultures can be used as an important strategy in studying the biologic mechanisms of TU action.
CONCLUSIONS We have provided novel evidence indicating that TU enhances the differentiation of skeletal muscle cells. However, more research is needed to explore the molecular and cellular basis of the observed TU effects during skeletal muscle differentiation. These results could have an impact on the development of new strategies and protocols for practical TU treatments.
Acknowledgments—This work was supported by Brazilian grants from the Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq), Programa de Capacitac¸~ao Cientıfica e Industrial do InmetroCNPq-PROMETRO No. 563089/2010-5, the Fundac¸~ao Carlos Chagas Filho de Apoio a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and the Programas de Apoio aos Nucleos de Excel^encia (Pronex). We thank Juliana Lourenc¸o Abrantes for technical assistance.
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REFERENCES Alvarenga AV, Costa-Felix R. Uncertainty assessment of effective radiation area and beam non-uniformity ratio of ultrasound transducers determined according to IEC 61689:2007. Metrologia 2009;46:367–374. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 1976;72:248–254. Chan YS, Hsu KY, Kuo CH, Lee SD, Chen SC, Chen WJ, Ueng SW. Using low-intensity pulsed ultrasound to improve muscle healing after laceration injury: An in vitro and in vivo study. Ultrasound Med Biol 2010;36:743–751. Dyson M. Mechanisms involved in therapeutic ultrasound. Physiotherapy 1987;73:116–120. Fisher BD, Hiller CM, Rennier SGA. A comparison of continuous ultrasound and pulsed ultrasound on soft tissue injury markers in the rat. J Phys Ther Sci 2003;15:65–70. Holtzer H, Dillulo C, Costa ML, Lu M, Choi J, Mermelstein CS, Scultheiss T, Holtzer S. Striated myoblasts and multinucleated myotubes induced in non-muscle cells by MyoD are similar to normal in vivo and in vitro counterparts. In: Ozawa E, Masaki T, Nabeshima Y, (eds). Frontiers in muscle research. Amsterdam: Elsevier; 1991. p. 187–207. Ikeda K, Takayama T, Suzuki N, Shimada K, Otsuka K, Ito K. Effects of low-intensity pulsed ultrasound on the differentiation of C2C12 cells. Life Sci 2006;79:1936–1943. Khanna A, Nelmes RT, Gougoulias N, Maffulli N, Gray J. The effects of LIPUS on soft-tissue healing: A review of literature. Br Med Bull 2008;89:169–182. Li Y, Wang J, Satterle A, Wu Q, Wang J, Liu F. Gene transfer to skeletal muscle by site-specific delivery of electroporation and ultrasound. Biochem Biophys Res Commun 2012;424:203–207. McBrier NM, Lekan JM, Druhan LJ, Devor ST, Merrick MA. Therapeutic ultrasound decreases mechano-growth factor messenger ribonucleic acid expression after muscle contusion injury. Arch Phys Med Rehabil 2007;88:936–940. Mermelstein CS, Portilho DM, Medeiros RB, Matos AR, Einicker-Lamas M, Tortelote GG, Vieyra A, Costa ML. Cholesterol depletion by methyl-b-cyclodextrin enhances myoblast fusion and
Volume 40, Number 3, 2014 induces the formation of myotubes with disorganized nuclei. Cell Tissue Res 2005;319:289–297. Nagata K, Nakamura T, Fujihara S, Tanaka E. Ultrasound modulates the inflammatory response and promotes muscle regeneration in injured muscles. Ann Biomed Eng 2013;41:1095–1105. Park H, Yip MC, Chertok B, Kost J, Kobler JB, Langer R, Zeitels SM. Indirect low-intensity ultrasonic stimulation for tissue engineering. J Tissue Eng 2010;1:1–9. Plentz RDM, Stoffel PB, Kolling GJ. Hematological changes produced by 1 MHz continuous ultrasound, applied during the acute phase of iatrogenic muscle injury in rats. Rev Bras Fisioter 2008;12: 495–501. Rantanen J, Thorsson O, Wollmer P, Hurme T, Kalimo H. Effects of therapeutic ultrasound on the regeneration of skeletal myofibers after experimental muscle injury. Am J Sports Med 1999;27:54–59. Robertson VJ, Baker KG. A review of therapeutic ultrasound: Effectiveness studies. Phys Ther 2001;81:1339–1350. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012;9:671–675. Schuster A, Schwab T, Bischof M, Klotz M, Lemor R, Degel C, Sch€afer KH. Cell specific ultrasound effects are dose and frequency dependent. Ann Anat 2013;195:57–67. Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, Currens MJ, Seniff D, Boyd MR. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 1988; 48:4827–4833. Shaw A, Hodnett M. Calibration and measurement issues for therapeutic ultrasound. Ultrasonics 2008;48:234–252. Silveira PC, da Silva LA, Tromm PT, Scheffer Dda L, de Souza CT, Pinho RA. Effects of therapeutic pulsed ultrasound and dimethylsulfoxide phonophoresis on oxidative stress parameters after injury induced by eccentric exercise. Ultrasonics 2012;52:650–654. Speed CA. Therapeutic ultrasound in soft tissue lesions. Rheumatology 2001;40:1331–1336. Ter Haar G, Shaw A, Pye S, Ward B, Bottomley F, Nolan R, Coady AM. Guidance on reporting ultrasound exposure conditions for bioeffects studies. Ultrasound Med Biol 2011;37:177–183. Watson T. Ultrasound in contemporary physiotherapy practice. Ultrasonics 2008;48:321–329.
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Original Contribution INDUCTION OF SKELETAL MUSCLE DIFFERENTIATION IN VITRO BY THERAPEUTIC ULTRASOUND VIVIANE MENDES ABRUNHOSA,*y CAROLINA PONTES SOARES,y ANA CLAUDIA BATISTA POSSIDONIO,y ANDRE VICTOR ALVARENGA,* RODRIGO P. B. COSTA-FELIX,* MANOEL LUIS COSTA,y and CLAUDIA MERMELSTEINy * Laboratorio de Ultrassom, Diretoria de Metrologia Cientıfica e Industrial (DIMCI), Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMETRO), Rio de Janeiro, RJ, Brazil; and y Instituto de Ci^encias Biomedicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil (Received 17 July 2013; revised 11 October 2013; in final form 15 October 2013)
Abstract—Therapeutic ultrasound (TU) has been used for the last 50 y in rehabilitation, including treatment of soft tissues. Ultrasound waves can be employed in two different modes of operation, continuous and pulsed, which produce both thermal and non-thermal effects. Despite the large-scale use of TU, there are few scientific studies on its biologic effects during skeletal muscle differentiation. To better analyze the cellular effects of TU, we decided to follow cells in vitro. The main purpose of this study was to evaluate the effects of TU in primary chick myogenic cell cultures using phase contrast optical microscopy and immunofluorescence microscopy, followed by image analysis and quantification. Our results indicate that TU can stimulate the differentiation of skeletal muscle cells in vitro, as measured by the thickness of multinucleated myotubes, the ratio of mononucleated cells to multinucleated cells and expression of the muscle-specific protein desmin. This study is a first step toward a metrologic and science-based protocol for cell treatment under different ultrasound field exposures. (E-mail: [email protected]) Ó 2014 World Federation for Ultrasound in Medicine & Biology. Key Words: Therapeutic ultrasound, Skeletal muscle differentiation, Desmin, Myogenesis.
or non-thermal (pulsed mode) (Dyson 1987). Thermal effects in vivo are derived from the conversion of ultrasonic energy into elevation of the tissue temperature between 40 C and 45 C for at least 5 min. These effects have been related to increased blood flow and flexibility of collagen fibers, reduction in muscle spasm and a proinflammatory response (Speed 2001). Non-thermal effects are the local vibrations that involve stable acoustic streaming and cavitations in tissue fluids and could improve tissue healing (Chan et al. 2010). Both effects, thermal and non-thermal, occur simultaneously during TU treatment. The administration of TU involves the choice of frequency, mode of operation of the equipment, effective intensity levels and duration of treatment. TU is broadly used in the frequency range of 0.5 to 5.0 MHz, but typically TU equipment can be set only at 1 or 3 MHz. Different frequencies are recommended depending on the injury and subcutaneous fat tissue of the patient. Frequencies of 1 MHz are absorbed primarily by tissues at a depth of 3–5 cm, and frequencies of 3 MHz are absorbed at a depth of 1–2 cm. The choice of mode of operation of
INTRODUCTION Soft tissue lesions are common in athletic and nonathletic populations and can be caused by direct contusions, indirect strain injuries or intensive exercise. These injuries affect muscle function, joint mobility and, thereby, quality of life (Rantanen et al. 1999). One of the most recommended treatments for a wide variety of skeletal muscle tissue injuries is therapeutic ultrasound (TU) (Speed 2001). Therapeutic applications of ultrasound have been widely used in physiotherapy since the 1950s (Shaw and Hodnett 2008; Watson 2008). Ultrasound is a form of mechanical energy with a highfrequency pressure wave that can be transmitted into the body (Khanna et al. 2008). The interaction of the ultrasound wave with the tissue can promote biophysical effects that can be classified as thermal (continuous mode)
Address correspondence to: Claudia Mermelstein, Universidade Federal do Rio de Janeiro, Instituto de Ci^encias Biomedicas, Av. Carlos Chagas Filho 373, Ilha do Fund~ao, 21941-902 Rio de Janeiro, RJ, Brazil. E-mail: [email protected] 504
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the equipment depends on the goal of the treatment. Continuous mode could cause a larger temperature increase in the tissue than pulsed mode, with other parameters being held constant. Although the mode of operation is related to temperature in the tissue, the intensity level is also involved. The effective intensity (W/cm2) is acquired from the quotient of maximum ultrasonic output power, delivered through the patient’s body, and the transducer’s effective radiating area. Dyson (1987) found that elevation of temperature in the tissue occurs in both continuous and pulsed mode. The other parameter involved in TU is the duration of treatment. There is no consensus on the duration of TU treatment, which can vary between 2 and 15 min for treatment of disorders in soft tissues. The amount of energy delivered to the tissue is proportional to the duration of treatment (Robertson and Baker 2001). The studies that have examined the effects of TU in soft tissues reported divergent results with respect to the efficacy of TU treatment. The variability in the efficacy of TU treatment could be caused by the lack of standardization in TU protocols and/or by the fact that different biologic models were used (Fisher et al. 2003; McBrier et al. 2007; Silveira et al. 2012). Although some of these studies were conducted in animal models, most were performed using the C2C12 mouse myoblast cell line. To better analyze the effects of TU at the cellular level, we studied primary skeletal muscle cell culture. Primary cultures of skeletal muscle cells are more similar to the in vivo system than is any muscle cell lineage. Thus, our main goal was to investigate the relationship between the protocols commonly used for TU treatment for muscle lesion recovery and their effects on skeletal muscle differentiation. The experiments were performed in primary cultures of chick skeletal muscle cells using TU equipment widely employed in physiotherapy. The chick myoblast primary culture is a robust in vitro model of myogenesis. Skeletal myogenesis proceeds in these cell cultures through the following main sequential stages: myoblast proliferation, cell cycle withdrawal, myoblast alignment, and fusion and their subsequent differentiation into striated multinucleated myotubes. The fact that these steps have been well characterized makes this an interesting model for analysis of the effects of TU during muscle differentiation. Regeneration of an injured skeletal muscle involves the activation of the proliferation of satellite cells, followed by their differentiation and fusion into myotubes, which contributes to the functional recovery of the muscle tissue. Differentiation of satellite cells recapitulates the embryonic myogenesis. The molecular and cellular basis of satellite cell differentiation can therefore be studied in embryonic myoblast cell culture models. One of the aims of our study was to determine whether TU treat-
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ment can activate the proliferation and differentiation of embryonic myoblasts, assuming that this process would be equivalent in muscle injury-activated satellite cells, and thus contribute to the comprehension of TU effects on muscle lesion healing. Our results indicated that skeletal muscle cell differentiation is induced in vitro by therapeutic ultrasound. METHODS Antibodies and fluorescent probes The DNA-binding probe 4,6-diamidino-2phenylindole dihydrochloride (DAPI), Alexa Fluor 488conjugated goat anti-mouse serum and Alexa Fluor 546-conjugated goat anti-rabbit serum were purchased from Molecular Probes (Eugene, OR, USA). Rabbit polyclonal anti-desmin (D-8281), mouse monoclonal antisarcomeric a-actinin (clone EA-53, A-7811) and mouse monoclonal anti-a-tubulin (clone DM1A, T-9026) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrasonic pressure field mapping system A water bath measuring 1700 3 1000 3 800 mm from Ultrasound Labs (National Institute of Metrology, Quality and Technology, Inmetro, Rio de Janeiro, Brazil) was used for mapping the ultrasound pressure field of the transducer. The specified positioning system (Newport, Irvine, CA, USA) used to move the transducer (or hydrophone) in the water bath allows movements of 300 mm along the x- and y-axes and 600 mm along the z-axis. The transducer is excited using a 20-cycle burst of sine wave generated by a function generator (AFG 3252, Tektronix, Beaverton, OR, USA), and waterborne signals are acquired using an oscilloscope (TDS 3032B, Tektronix). In the mapping procedure, a needle hydrophone is used with an active element of 0.2 mm (Precision Acoustics, Dorchester, Dorset, UK). To integrate all system components, and also to provide a user-friendly interface, virtual software was developed in the LabView platform (National Instruments, Austin, TX, USA). The software automatically performs the raster scan necessary to calculate the effective radiating area (AER) of the physiotherapeutic ultrasound transducer (Alvarenga and Costa-Felix 2009). Primary myogenic cell culture This study using chick embryos was approved by the Ethics Committee for Animal Care and Use in Scientific Research of the Federal University of Rio de Janeiro and received Approval No. DAHEICB004. All cell culture reagents were purchased from Invitrogen (S~ao Paulo, Brazil). Primary cultures of myogenic cells were prepared from breast muscles of 11-d-old chick embryos (Mermelstein et al. 2005). Cells were plated at an initial density of 106 cells/35 mm in culture dishes coated with
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rat tail collagen. Cells were grown with 2 mL of culture medium (Minimum Essential Medium supplemented with 10% horse serum, 0.5% chick embryo extract, 1% L-glutamine, and 1% penicillin-streptomycin) under a humidified 5% CO2 atmosphere at 37 C. Twenty-fourhour cultures were treated with therapeutic ultrasound waves (see Table 1). Sodium bicarbonate (1.4 g/L) was added to the culture medium to maintain pH in the range of 7.2–7.4. Phenol red, a pH indicator, was added to medium to colorimetrically monitor changes in pH in both untreated and ultrasound-treated cells. After treatment, cultures were grown for the next 48 h and culture medium was changed every day. Treatment with ultrasound wave An ultrasound device (Model Sonopulse Compact, Ibramed, Amparo, Brazil) was used in different modes of operation (continuous or pulsed) at different effective intensities (0.5 or 1 W/cm2) for different durations of treatment (5 or 10 min). The frequency of the TU was the same for all treatments (1 MHz). Ultrasound was transmitted through the bottom of the culture dish, with a coupling gel between the ultrasound transducer and the dish. The ultrasonic attenuation effects within the medium were disregarded, as they were the same throughout all cell cultures and different treatments, and the free path is markedly short (about 2 mm). Most likely, reflections on the free surface of the medium will result in an identical ultrasonic field inside the medium, as all culture dishes were prepared in a similar way with respect to the amount (volume) of medium. Both attenuation and surface reflections were considered inherent characteristics of the ultrasonic pressure field and very similar to each other in all tested cultures. The diameter of the ultrasound transducer was 35 mm (the same as the culture dish). All 24-h cell cultures were exposed to TU treatment only once. Control cultures were subjected to the same experimental procedures as the treated cultures, except that the ultrasound Table 1. Protocols for ultrasound wave treatment* Mode of operation Continuous Continuous Continuous Continuous Pulsed Pulsed Pulsed Pulsed
Intensity (W/cm2)
Duration of treatment (min)
0.5 0.5 1.0 1.0 0.5 0.5 1.0 1.0
5 10 5 10 5 10 5 10
* An ultrasound apparatus was used in different modes of operation (continuous or pulsed) at different effective intensities (0.5 or 1 W/ cm2) for different durations of treatment (5 or 10 min). The frequency of therapeutic ultrasound was the same for all treatments (1 MHz).
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equipment was not turned on (sham ultrasound). The different TU treatments used in cells are described in Table 1. All analyses were performed 48 h after treatment (which means that the cells were grown in culture for a total of 72 h). The ultrasound apparatus was certificated according to IEC 61689:2007 before its use in this study. The temperature of the culture medium was measured immediately before and after TU treatment. Temperature was measured using an infrared thermometer (Model 62MAX1, Fluke, Beijing, China) directly on the medium of each dish without the cover lid. Cell viability Cell viability was measured with the XTT assay (Scudiero et al. 1988). XTT (2,3-bis (2-methoxy-4-nitro5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide, Sigma-Aldrich, USA) is metabolically reduced in viable cells to a water-soluble formazan product. This reagent allows direct absorbance readings. XTT was prepared at 1 mg/mL in pre-warmed (37 C) Minimum Essential Medium without serum. PMS (phenazine methosulfate, Sigma-Aldrich) was prepared at 5 mM (1.53 mg/mL) in phosphate-buffered solution (PBS). Fresh XTT and PMS were mixed together in the appropriate concentrations. For a 0.025 mM PMS-XTT solution, 25 mL of the stock 5 mM PMS was added per 5 mL of XTT (1 mg/mL). One hundred microliters of this mixture (final concentration, 50 mg XTT and 0.38 mg PMS per well) was added to each of the 96 wells on the plate containing 24-h myogenic cell cultures (untreated and TU-treated). After incubation at 37 C for 24 h, the absorbance at 492 nm was measured with a SunriseBasic spectrophotometer (Tecan, Gr€odig, Austria). Immunofluorescence microscopy and digital image acquisition Myogenic cells were rinsed with PBS for 10 min at room temperature. Cells were then permeabilized with 0.5% Triton-X 100 in PBS three times for 10 min. The same solution was used for all subsequent washing steps. Cells were incubated with primary antibodies for 1 h at 37 C in a humidity chamber. After incubation, cells were washed for 30 min and incubated with Alexa Fluor 488 and/or Alexa Fluor 546-conjugated secondary antibodies for 1 h at 37 C in a humidity chamber. After a 10-min wash with 0.9% NaCl, DAPI (0.1 mg/mL in 0.9% NaCl) was added for 5 min. Cells were mounted in ProLong Gold anti-fade reagent (Molecular Probes) and examined with an Axiovert 100 microscope (Carl Zeiss, Jena, Germany) by using filter sets that were selective for each fluorochrome wavelength channel. Images were acquired with a DP72 camera (Olympus, Tokyo, Japan). All nuclei labeled with DAPI were counted in mononucleated and multinucleated desmin-positive cells.
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Quantification of cultured images Phase contrast microscopy images of live cultured cells were acquired with an Axiovert 100 microscope (Carl Zeiss). Images of 72-h myogenic cultures were acquired from several equidistant microscopic fields, representative of the whole dish for each culture condition. Diameter of myotubes and number of nuclei were obtained using the public domain software ImageJ (Schneider et al. 2012). Myotube thickness was measured in the thicker region of each myotube, for all myotubes in each microscopic field, in at least 50 different fields for each experimental condition. The data were collected from three independent experiments.
Statistical analysis Data are presented as the mean 6 standard deviation. Mean values were calculated from at least three independent experiments. Tests of statistical significance were performed using IBM SPSS software. Statistical analysis of the data related to myotube thickness was performed by one-way analysis of variance. An unpaired t-test was used for the data related to the nucleus quantification. Statistical comparisons of the immunoblot data were performed with Student’s t-test. The values were considered to be statistically different at p-values # 0.05.
SDS-PAGE and immunoblotting Untreated and TU-treated myogenic cultures were grown for 72 h. Cultures were then quickly washed in ice-cold PBS. Fifty microliters of ice-cold sample buffer (4% sodium dodecyl sulfate [SDS], 20% glycerol, 0.2 M dithiothreitol, 125 mM Tris-HCl, pH 6.8) was added to the cultures, and then cells were scraped off the dish with a plastic cell scraper. Cell extracts were recovered in a tube, boiled for 5 min at 95 C and centrifuged at 12,000g for 15 min. The pellet was discarded and the supernatant was used in subsequent steps. The amount of protein in each sample was determined according to the Bradford (1976) method, using bovine serum albumin as a standard. Equal amounts of protein were loaded on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to poly (vinyl difluoride) membranes. The proteins immobilized on the membranes were immediately blocked for 1 h at room temperature with 5% non-fat dry milk in Tris-buffered saline-Tween 20 solution (0.001%) (TBS-T). Then the membranes were incubated for 12 h at 37 C with an anti-desmin polyclonal antibody. After five washes in TBS-T (3 min each), the membranes were incubated for 1 h at 37 C with an anti-rabbit peroxidase-conjugated antibody (dilution 1:10,000 in TBS-T; Amersham, GE Healthcare, Pittsburgh, PA, USA) and washed again as described above; the bands were visualized using the ECL Plus Western Blotting Detection System (Amersham/GE Healthcare). To check sample loading, other membranes with the same samples were incubated with a mouse monoclonal anti-a-tubulin antibody (Sigma-Aldrich, dilution 1:3000 in TBS-T-milk). After five washes in TBS-T (3 min each), membranes were incubated with anti-mouse peroxidase-conjugated antibody (Amersham, dilution 1:10,000 in TBS-T) and developed as described above. Quantification of protein bands was performed using the public domain software ImageJ (Schneider et al. 2012) with data obtained from three independent experiments.
Therapeutic ultrasound transducer reveals a non-homogeneous pressure field First, we established the effectiveness of the ultrasound system used by evaluating the ultrasound pressure field of the transducer by mapping the ultrasound field in an acoustic tank. The transducer is non-focused; that is, it has a natural focus. Its last maximum of ultrasonic pressure amplitude was measured at 109.5 6 2.7 mm from its surface. The transducer’s mapping disclosed non-homogeneity over its output surface, but the nonuniformity ratio of the ultrasonic beam was less than 8. It leads to non-uniform output, but without marked hot spots. Moreover, the medium depth is short compared with the transducer diameter (2 and 35 mm, respectively), which results in near-field behavior throughout the medium. In this case, diffraction interference is not expected to prevail, despite the multiple reflections and attenuation that might occur. The average value of AER for the physiotherapy transducer used in this study was 2.43 cm2, with an estimated expanded uncertainty of 2.9 3 10 cm2
RESULTS
Fig. 1. Cell viability of untreated and therapeutic ultrasound (TU)-treated myogenic cell cultures. Myogenic cells were grown for 24 h, subjected to sham ultrasound (control) or different TU treatments and grown for the next 24 h. Cell viability was quantified by the XTT assay (Scudiero et al. 1988) assay in three independent experiments. Note that cell viability did not change significantly after ultrasound treatment. Treatment parameters are given as continuous (cont) or pulsed (pul) mode–intensity (W/cm2)–duration (min).
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Fig. 3. Mean thickness of myotubes in untreated and therapeutic ultrasound (TU)-treated myogenic cell cultures. Myogenic cells were grown for 24 h, subjected to sham ultrasound (control) or different TU treatments and grown for the next 48 h. Myotube thickness was measured in at least 50 different microscopic fields for each experimental condition from three independent experiments. Note that some TU treatments induced significant enhancement in the thickness of myotubes. *p , 0.05; one-way analysis of variance, compared with control, n 5 3. Treatment parameters are given as continuous (cont) or pulsed (pul) mode–intensity (W/cm2)–duration (min).
guideline, this study was framed in level 1, because the results were referred to the physiotherapy system’s nominal ultrasonic intensity instead of the actual acoustic output.
Fig. 2. Phase contrast microscopy analysis of untreated and therapeutic ultrasound (TU)-treated myogenic cell cultures. Myogenic cells were grown for 24 h, subjected to sham ultrasound (control) or different TU treatments and grown for the next 48 h. Experimental conditions are detailed in Table 1. One myotube in each image is in green. The yellow line in the image of cells treated with continuous mode ultrasound at 0.5 W/cm2 for 5 min indicates how myotube thickness was measured. Treatment parameters are given as continuous (cont) or pulsed (pul) mode– intensity (W/cm2)–duration (min). Bar 5 50 mm.
(11.9%). To understand the relationship between exposure conditions and any observed biologic effects, a guideline was proposed by ter Haar et al. (2011). According to this
TU does not affect cell viability in myogenic cultures Next, we tested the effects of the continuous and pulsed modes of operation, two different intensities (0.5 and 1.0 W/cm2) and two different durations of treatment (5 and 10 min) in a primary culture of chick embryonic skeletal muscle cells. The treatments tested here were chosen by taking into consideration the protocols most commonly used in clinical practice. To determine whether TU treatment has any effect on cell viability, experiments were performed with untreated and TU-treated myogenic cultures grown 24 h after TU exposure, using a XTT-based method (Scudiero et al. 1988). No significant effect on cell viability was noted in the myogenic cells 24 h after TU treatment compared with untreated cells (Fig. 1). We also measured the temperature of the culture medium after TU treatment and did not observe an increase in temperature. TU induces differentiation of embryonic skeletal muscle cells Phase contrast microscopy revealed that some TU treatments enhanced muscle differentiation, as suggested by an increase in myotube thickness (Fig. 2). TU
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Fig. 4. a-Actinin distribution in untreated and therapeutic ultrasound (TU)-treated myogenic cell cultures. Myogenic cells were grown for 24 h, subjected to sham ultrasound (a, c) or continuous ultrasound mode at 0.5 W/cm2 for 5 min (cont–0.5–5) and grown for the next 48 h (b, d). Cells were fixed and double-stained with an anti-sarcomeric a-actinin antibody (green, a–d), and the nuclear dye 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue, a and b). A merged image is provided in (a) and (b). Note that cultures treated with cont–0.5–5 (d) had thicker myotubes than untreated cells (c). a-Actinin (in green) in localized in the Z-bands of striated myofibrils in both untreated and ultrasound-treated myotubes. Myotube thickness is represented by white line in (d). Bar 5 100 mm (a, b) and 20 mm (c, d).
treatment in continuous mode at 0.5 W/cm2 for 5 or 10 min (cont–0.5–5 and cont–0.5–10), TU in continuous mode at 1.0 W/cm2 for 5 min (cont–1.0–5) and TU in pulsed mode at 1.0 W/cm2 for 5 min (pul–1.0–5) differed statistically significantly (95% confidence interval), as measured by myotube thickness, compared with no treatment (Fig. 3). To confirm whether or not the cont–0.5–5 treatment enhanced muscle differentiation, we analyzed the expression and distribution of the muscle-specific marker a-actinin. We decided to test only the cont–0.5–5 treatment because our previous results (Figs. 2 and 3) had indicated that this TU treatment promoted major changes in myotube thickness. a-Actinin is a myofibrillar protein that localizes in Z-bands in sarcomeres. Immunofluorescence images revealed the presence of a-actinin in striated myofibrils in multinucleated myotubes in both untreated and ultrasound-treated cultures (Fig. 4). Cell cultures subjected to cont–0.5–5 displayed more and thicker myotubes, confirming the enhancement in skeletal muscle differentiation. It is possible to observe a larger number of nuclei (labeled with the nuclear dye DAPI) in the TU-treated condition (Fig. 4a, b), which could be inter-
preted as an increase in cell proliferation after TU exposure. These results indicated once again that cont–0.5–5 induced the formation of thicker myotubes compared with no treatment. We also quantified the number of nuclei in mononucleated and multinucleated (myotubes) cells in untreated cultures and cultures treated with cont–0.5–5. Our results indicate that cont–0.5–5 induced a 30% increase in the total number of nuclei per field and a 60% increase in the total number of nuclei per myotube (Fig. 5). Note also that the proportion of nuclei in mononucleated cells, compared with nuclei in myotubes, is even higher in TU-treated cultures (from 1.5 to 3.1). The increase in the total number of cells in TU-treated cultures could indicate enhancement of cell proliferation and/or survival. The increase in the ratio of mononucleated cells to multinucleated cells suggests enhancement of cell fusion. Next we decided to quantify the amount of desmin protein expression in untreated cells and cells treated with cont–0.5–5. Cells were grown for 24 h, treated with TU and grown for the next 48 h. Untreated and TUtreated cell extracts were analyzed by 10% SDS-PAGE
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Fig. 5. Average number of nuclei in untreated and therapeutic ultrasound (TU)-treated myogenic cell cultures. Myogenic cells were grown for 24 h, subjected to sham ultrasound (control) or continuous ultrasound mode at 0.5 W/cm2 for 5 min (cont–0.5–5) and grown for the next 48 h. The nuclei present in mononucleated cells and multinucleated cells (myotubes) were quantified in at least 50 different microscopic fields for each experimental condition from three independent experiments. Note that cont–0.5–5 induced a 30% increase in the total number of nuclei per field and a 60% increase in the total number of nuclei per myotube. *p , 0.05, unpaired t-test, compared with control, n 5 3.
followed by immunoblotting. Quantification of immunoblots revealed a 60% increase in the expression of desmin (52 kDa) in TU-treated cells compared with control cells (Fig. 6). DISCUSSION In the work described here, we studied the effects of commonly used TU treatment protocols for muscle lesion recovery in chick skeletal muscle cell culture to determine the relationship between TU protocols and their effects on muscle differentiation. We found that several of these protocols, most of them in continuous mode, enhance skeletal muscle fiber formation. Muscle differentiation was reflected in the presence of more myotubes with more well-organized striated myofibrils. The extent of differentiation was analyzed by measurement of the thickness of multinucleated myotubes and the ratio of mononucleated cells to multinucleated cells. These findings argue that the chick in vitro model of myoblast cell culture is a valuable tool for studies of the cellular and molecular effects of TU during muscle differentiation. In vitro models such as primary chick myogenic culture begin with a population of replicating mononucleated myoblasts and some fibroblastic cells (Holtzer et al. 1991). After a few rounds of replication, these myoblasts withdraw from the cell cycle and begin to change in
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Fig. 6. Therapeutic ultrasound (TU) enhances the expression of desmin in myogenic cell cultures. (a) Total cell extracts from primary cultures of chicken myogenic cells were analyzed by Western blotting using an anti-desmin antibody. Cells were grown for 24 h, subjected to sham ultrasound (control) or continuous ultrasound mode at 0.5 W/cm2 for 5 min (cont–0.5–5) and grown for the next 48 h. (b) Quantification of immunoblots revealed a 60% increase in the levels of desmin expression (52 kDa) in cells after TU treatment compared with control cells. The semi-quantitative analysis represents the average of three independent experiments. *p , 0.05, Student’s t-test, compared with control, n 5 3.
shape, becoming elongated and bipolar cells. Bipolar myoblasts then enter a cell-cell recognition phase in which their plasma membranes adhere and fuse. Fusion gives rise to multinucleated and striated myotubes. The enhancement in thickness of myotubes and number of nuclei per myotube after TU treatment could be caused by an increase in myoblast fusion. Myoblast fusion can be enhanced in different ways, such as an increase in membrane fluidity and/or permeability, an increase in myoblast proliferation (which occurs before fusion) and exposure or removal of membrane molecules that are necessary for the fusion process. It has been proposed that TU treatment induces changes in cell membrane permeability, a property that is widely used for improving gene transfer protocols (Li et al. 2012). Few studies have examined the effect of TU in the treatment of skeletal muscle (Fisher et al. 2003; McBrier et al. 2007; Plentz et al. 2008), and none of them found significant differences using continuous mode treatment. Our results contradict those articles, because we found that all continuous mode TU treatments, with the exception of cont–1.0–10, induced
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enhancement of myotube thickness. Several explanations for these contradictory findings can be hypothesized. One possible explanation might be the difference between their in vivo approach and our in vitro approach. Fewer variables are involved in myogenesis in skeletal muscle cell cultures than in animal myogenesis. In an animal, there are hundreds of cell types interacting with each other and with the extracellular matrix, through the exchange of chemical signals in a 3-D environment. In contrast, cell cultures respond to a reduced range of stimuli in a controlled environment. Therefore, cell culture models are usually less variable and provide higher reproducibility than animal models. In addition, the results obtained in cell cultures are produced directly by the cells and their controlled environment, whereas in animals, the results could arise from complex interactions between tissues and/or systems. Other possible explanations for the contradictory findings between our results and those of other authors are: (i) differences between species (birds vs. mammals); (ii) differences between a muscle damage recovery model and our normal myogenesis model; and (iii) differences in TU conditions used, such as, frequency, intensity of the equipment and duration of treatment, as suggested by Schuster et al. (2013). The enhancement in myogenesis we observed is probably not related to the thermal effect of the continuous or pulsed mode condition used, as no increase in temperature was detected in the myogenic cell cultures after TU treatments. Most of the cell cultures we treated with pulsed TU did not exhibit an increase in myotube thickness, with the exception of pul–1.0–5, whereas myotube thickness increased in most continuous TU cultures. This finding may be explained by the fact that the energy delivered to the culture by pulsed mode at 1.0 W/cm2 is the same as that delivered by continuous mode at 0.5 W/cm2 during the 5 min in which we observed positive effects on muscle thickness. Interestingly, we could not observe any correlation between the overall energy delivered by TU to cells and their differentiation. Our results are in agreement with those of Fisher et al. (2003), who reported a significant increase in synthesis of contractile proteins in injured gastrocnemius of male Sprague-Dawley rats after treatment with the same of mode of operation (pulsed), intensity (1.0 W/cm2) and duration of treatment (5 min) used in our study. Studies using TU conditions similar to those described above have suggested that these results are relative to an increase in satellite cell proliferation (Rantanen et al. 1999). In contrast to our results, Ikeda et al. (2006) reported that low-intensity pulsed ultrasound (LIPUS, 1.5 MHz, 70 mW/cm2 for 20 min) induces a significant decrease in expression of the myogenic transcription factor
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MyoD in the myoblast cell line C2C12. In agreement with our data, Nagata et al. (2013) described that treatment of C2C12 cells with TU (LIPUS, 3 MHz, 30 mW/ cm2 for 15 min) up-regulated expression of the myogenic transcription factor myogenin, and treatment of mouse muscle with TU induced an increase in the crosssectional area of myofibers. Park et al. (2010) reported that C2C12 cells subjected to LIPUS (30 mW/cm2 for 3 or 10 min daily for 6 d) exhibited a significant increase in expression of tropomyosin. Thus, we can conclude from the results presented here with primary cultures of chick myoblasts and from the data previously described by other groups with the C2C12 cell line (Nagata et al. 2013; Park et al. 2010) that some TU treatments can promote myogenesis and, thus, can be a valuable tool in treatment of muscle injuries. It is difficult to draw further conclusions from these studies because of differences in the test systems. Furthermore, inadequate calibration of TU equipment can also interfere with treatment. It has been reported that performance of TU equipment that is more than 10 y old and equipped with dual-frequency treatment heads can be compromised (Speed 2001). To avoid these problems, we sought to certify our TU equipment. We acknowledge the differences between treating muscle cells in vitro with TU and treating human muscle tissues in vivo. In addition to the fact that cell cultures lacks the complexity of muscle tissues, as mentioned above, there is also the problem of artificial interactions between the acoustic field and the plastic culture dish. In the present work, we used a coupling gel between the TU transducer and the plastic culture dish to minimize these unwanted interactions. On the other hand, as we illustrated the direct effects of TU on cell differentiation, cell cultures can be used as an important strategy in studying the biologic mechanisms of TU action.
CONCLUSIONS We have provided novel evidence indicating that TU enhances the differentiation of skeletal muscle cells. However, more research is needed to explore the molecular and cellular basis of the observed TU effects during skeletal muscle differentiation. These results could have an impact on the development of new strategies and protocols for practical TU treatments.
Acknowledgments—This work was supported by Brazilian grants from the Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq), Programa de Capacitac¸~ao Cientıfica e Industrial do InmetroCNPq-PROMETRO No. 563089/2010-5, the Fundac¸~ao Carlos Chagas Filho de Apoio a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and the Programas de Apoio aos Nucleos de Excel^encia (Pronex). We thank Juliana Lourenc¸o Abrantes for technical assistance.
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