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The use of defined in vitro systems to study the developmental and physiological characteristics of a variety of cell types is increasing, due in large part to their ease of integration with tissue engineering, regenerative medicine, and high-throughput screening applications. In this study, myotubes derived from fetal rat hind limbs were induced to develop several aspects of mature muscle including: sarcomere assembly, development of the excitation-contraction coupling apparatus and myosin heavy chain (MHC) class switching. Utilizing immunocytochemical analysis, anisotropic and isotropic band formation (striations) within the myotubes was established, indicative of sarcomere formation. In addition, clusters of ryanodine receptors were colocalized with dihydropyridine complex proteins which signaled development of the excitation-contraction coupling apparatus and transverse tubule biogenesis. The myotubes also exhibited MHC class switching from embryonic to neonatal MHC. Lastly, the myotubes survived significantly longer in culture (70–90 days) than myotubes from our previously developed system (20–25 days). These results were achieved by modifying the culture timeline as well as the development of a new medium formulation. This defined model system for skeletal muscle maturation supports the goal of developing physiologically relevant muscle constructs for use in tissue engineering and regenerative medicine as well as for high-throughput screening applications.
Skeletal muscle differentiation and maturation is a complex process involving the synergy of different growth factors and hormones interacting over a broad time period [1–4]. The differentiation process is further complicated by neuronal innervation, where neuron to muscle cell signaling can regulate myosin heavy chain (MHC) gene expression and acetylcholine receptor clustering [5–7]. Consequently, understanding of the role of growth factors, hormones and cellular interactions in skeletal muscle differentiation would be a key step in generating physiologically relevant tissue engineered constructs, developing advanced strategies for regenerative medicine and integrating functional skeletal muscle with bio-hybrid MEMS devices for non-invasive interrogation in high-throughput screening technologies.
In order for skeletal muscle myotubes developed in vitro to be useful in tissue engineering applications, they must exhibit as many of the functional characteristics of in vivo skeletal muscle fibers as possible. During muscle fiber development in vivo, several critical structural changes occur that indicate functional maturation of the extrafusal myotubes. These changes include sarcomere organization, clustering and colocalization of ryanodine (RyR) and dihydropyridine (DHPR) receptors and MHC class switching [8, 9]. Each of these structural changes reflects the physiological maturation of the skeletal muscle and is critical for consistent muscular contraction. For example, organization of the contractile proteins myosin and actin into sarcomeric units gives skeletal muscle myotubes organized and structured contraction, a property lacking in smooth muscle. The organization of sarcomeres in skeletal muscle gives rise to anisotropic and isotropic bands of proteins (A and I bands) and gives skeletal muscle a striated appearance. The clustering and colocalization of RyR and DHPR is indicative of transverse tubule (T-tubule) biogenesis and excitation contraction coupling. This developmental step structurally links electrical excitation to the internal contractile system by providing close apposition of DHPR located in the T-tubule and RyR located in the sarcoplasmic reticulum. Finally, a properly functioning skeletal muscle must express the appropriate MHC proteins required for the task it must perform. For example, different muscle fibers express different MHC proteins depending on the rate of contraction and force generation required by the work to be done. Without these modifications, an in vitro model of skeletal muscle maturation cannot achieve full physiological relevance.
One approach for identifying the role of specific growth factors and hormones in muscle differentiation is to develop an in vitro model system consisting of a serum-free medium supplemented with the factors of interest . Such a model provides the opportunity to evaluate the role of each factor individually or in combination with others known or believed to be important in skeletal muscle development. It also has the benefit of removing factors that may be present in serum that would inhibit maturation. Employing a non-biological growth substrate such as trimethoxy-silylpropyl-diethylenetriamine (DETA) provides an additional measure of control. DETA is a silane molecule that forms a covalently bonded monolayer on glass coverslips, resulting in a uniform, non- hydrophilic surface for cell growth and may partially mimic the three dimensional features of an extracellular matrix, which may be responsible for the robust growth found for different cell types on this synthetic substrate [11–15]. Previously, studies have demonstrated the usefulness of the DETA silane substrate for in vitro culture systems [10, 16, 17]. The use of DETA surfaces is advantageous from a tissue engineering perspective because it can be covalently linked to virtually any hydroxolated surface, it is amenable to patterning using standard photolithography  and it promotes long-term cell survival because it is non-digestible by matrix metalloproteinases secreted by the cells [15, 18].
A defined system that promoted differentiation of different skeletal muscle phenotypes was developed earlier and resulted in the formation of contractile myotubes, but with only short-term survival [12, 15]. In a technological advance we have also developed a novel bio-hybrid system to integrate functional myotubes with cantilever based bio-MEMS devices for the study of muscle physiology, neuromuscular junction formation, bio-robotics applications and for use in a model of the stretch reflex arc . More recently, using our defined model system, we have achieved a significant breakthrough by creating mechanosensitive intrafusal myotubes in vitro . Intrafusal fibers are the myotubes present in the muscle spindle which functions as the sensory receptor of the stretch reflex circuit  and combined with extrafusal fibers represent the primary component necessary to reproduce functional muscle activity in vitro.
This system has been utilized as a model for different developmental and functional applications, however, further improvements were necessary to enhance the physiological relevance of the skeletal muscle myotubes [19, 20]. Specifically, in order to create a working model of the stretch reflex arc, myotubes are needed that more accurately represent extrafusal fibers in vivo. In this study, we have now demonstrated sarcomere assembly, the development of the excitation-contraction coupling apparatus and myosin heavy chain (MHC) class switching. These results suggest we have discovered a group of biomolecules that act as a molecular switch promoting the transition from embryonic to neonatal MHC expression as well as other structural adaptations resulting in the maturation of skeletal muscle in vitro. The discovery of these biomolecular switches will be a powerful tool in regenerative medicine and tissue engineering applications such as skeletal muscle tissue grafts. It should also be useful in the creation of higher content high-throughput screening technologies.
Glass coverslips (Thomas Scientific 6661F52, 22 × 22mm No.1) were cleaned using an O2 plasma cleaner (Harrick PDC-32G) for 20 minutes at 100 mTorr. The DETA (United Chemical Technologies Inc. T2910KG) films were formed by the reaction of the cleaned glass surface with a 0.1% (v/v) mixture of the organosilane in freshly distilled toluene (Fisher T2904). The DETA coated coverslips were then heated to approximately 100°C, rinsed with toluene, reheated to approximately 100°C, and then oven dried . Surfaces were characterized by contact angle measurements using an optical contact angle goniometer (KSV Instruments, Cam 200) and by X-ray photoelectron spectroscopy (XPS) (Kratos Axis 165). XPS survey scans, as well as high-resolution N1s and C1s scans utilizing monochromatic Al Kα excitation were obtained .
Skeletal muscle was dissected from the thighs of the hind limbs of fetal rat (17–18 days old). The tissue was collected in a sterile 15 mL centrifuge tube containing 1 mL of phosphate-buffered saline (calcium- and magnesium-free) (Gibco 14200075). The tissue was enzymatically dissociated using 2 mL of 0.05% of trypsin-EDTA (Gibco 25300054) solution for 30 minutes in a 37°C water bath at 50 rpm. After 30 minutes the trypsin solution was removed and 4 mL of Hibernate E + 10% fetal bovine serum (Gibco 16000044) was added to terminate the trypsin reaction. The tissue was then mechanically triturated with the supernatant being transferred to a 15 mL centrifuge tube. The same process was repeated two times by adding 2 mL of L15 + 10% FBS each time. The 6 mL cell suspension obtained after mechanical trituration was suspended on a 2 mL, 4% BSA (Sigma A3059) (prepared in L15 medium) cushion and centrifuged at 300g for 10 minutes at 4°C. The pellet obtained was washed 5 times with L15 medium then resuspended in 10 mL of L15 and plated in 100 mm uncoated dishes for 30 minutes. The non-attached cells were removed and then centrifuged on a 4% BSA cushion . The pellet was resuspended in serum-free medium according to the protocol illustrated in Figure 1 and plated on the coverslips at a density of 700–1000 cells/mm2. The serum-free medium containing different growth factors and hormones was added to the culture dish after one hour. The final medium was prepared by mixing medium one (Table 1) and medium two (Table 2) in a 1:1 v/v ratio. Figure 1 indicates the flowchart of the culture protocol. Table 1 and Table 2 list the growth factor and hormone supplement compositions of medium one and medium two. The cells were maintained in a 5% CO2 incubator (relative humidity 85%). The full medium was replaced after four days with NBactiv4 medium according to the protocol in Figure 1 . Thereafter three-fourths of the medium was changed every three days with NBactiv4.
Coverslips were prepared for immunocytochemical analysis as previously described. Briefly, coverslips were rinsed with PBS, fixed in −20°C methanol for 5–7 min, washed in PBS, incubated in PBS supplemented with 1% BSA and 0.05% saponin (permeabilization solution) for 10 minutes, and blocked for 2h with 10% goat serum and 1% BSA. Cells were incubated overnight with primary antibodies against embryonic myosin heavy chain (F1.652) (dilution>1:5), neonatal myosin heavy chain (N3.36) (1:5) (Developmental Studies Hybridoma Bank), ryanodine receptor (AB9078, Millipore) (1:500) and dihydropyridine binding complex (α1-Subunit) (MAB 4270, Millipore) (1:500) diluted in the blocking solution. Cells were washed with PBS and incubated with the appropriate secondary antibodies for two hours in PBS. After two hours the coverslips were rinsed with PBS and mounted on glass slides and evaluated using confocal microscopy .
AChRs were labeled as described previously by incubating cultures with 5 × 10−8 M of α-bungarotoxin, Alexa Fluor® 488 conjugate (B-13422; Invitrogen) for 1.5 h at 37°C [5, 14]. Following incubation in α-bungarotoxin, the cultures were fixed as above for subsequent staining with embryonic myosin heavy chain (F1.652) antibodies.
Whole-cell patch clamp recordings were performed in a recording chamber located on the stage of a Zeiss Axioscope 2FS Plus upright microscope as described previously [15, 20]. The chamber was continuously perfused (2 ml/min) with the extracellular solution (Leibovitz medium, 35˚C). Patch pipettes were prepared from borosilicate glass (BF150-86-10; Sutter, Novato, CA) with a Sutter P97 pipette puller and filled with intracellular solution (K-gluconate 140 mM, EGTA 1 mM, MgCl2 2 mM, Na2ATP 2 mM, phosphocreatine 5mM, phosphocreatine kinase 2.4 mM, Hepes 10 mM; pH=7.2). The resistance of the electrodes was 6–8MΩ. Voltage clamp and current clamp experiments were performed with a Multiclamp 700A amplifier (Axon Laboratories, Union City, CA). Signals were filtered at 2 kHz and digitized at 20 kHz with an Axon Digidata 1322A interface. Data recording and analysis were done with pClamp 8 software (Axon Laboratories). Membrane potentials were corrected by subtraction of a 15 mV tip potential, which was calculated using Axon’s pClamp 8 program. Sodium and potassium currents were measured in voltage clamp mode using voltage steps from a −85 mV holding potential. Action potentials were evoked with 1 second depolarizing current injections from a − 85 mV holding potential [12, 15].
Static contact angle and XPS analysis was used for the validation of the surface modifications and for monitoring the quality of the surfaces. Stable contact angles (40.64° ± 2.9 /mean ± SD) throughout the study indicated high reproducibility and quality of the DETA surfaces and these characteristics were similar to previously published results [14, 15]. Based on the ratio of the N (401 and 399 eV) and the Si 2p3/2 peaks, XPS measurements indicated that a reaction-site limited monolayer of DETA was formed on the coverslips .
The serum free medium composition was developed semi-empirically. The final medium is derived from two different medium compositions described in Table 1 and Table 2. Table 1 constitutes the same medium composition used previously for a motoneuron-muscle co-culture and adult spinal cord neurons culture [11, 14]. Table 2 is composed of twelve additional factors that had been shown to promote skeletal muscle maturation and neuromuscular junction formation individually. The final medium was prepared by mixing these two mediums in a 1:1 v/v ratio. After the first 4 days of culture the whole medium was replaced with NBactiv4 medium . Thereafter, every three days three-fourths of the medium was changed with NBactiv4. The culture technique has been illustrated in the flowchart in Figure 1.
Using this new medium formulation and timeline, myotubes were successfully cultured for more than 90 days. Figure 2 indicates 50 days old myotubes in culture. As the myotubes aged and grew they began to form the characteristic anisotropic (A band) and isotropic (I band) banding pattern observed with mature in vivo muscle fibers [9, 23]. This banding pattern is caused by differential light diffraction due to the organization of myofibril proteins forming sarcomeres within the myotubes [9, 23]. The arrowheads in the images (Figure 2 A–D) indicate myotubes where sarcomeric organization has occurred and is visualized by the appearance of the A and I bands.
The myotubes formed were evaluated for the expression of fetal MHC to establish a baseline as comparison to our previous results . In Figure 3, the different myotube phenotypes formed at approximately day 50 in vitro are shown. The myotubes ranged from having clustered nuclei (Figure 3 A–D) to having diffuse nuclear organization (Figure 3 E–H). The arrowheads in the images indicate the characteristic striations.
In order to determine if the myotubes were maturing in a physiologically relevant way as they aged in vitro, the expression of neonatal MHC protein was evaluated. After approximately 50 days in vitro, 25% of the myotubes expressed neonatal MHC (Figure 4 A–M). Additionally, the myotubes were stained to detect the clustering of acetylcholine receptors (AChR) using alpha bungarotoxin (Figure 5 B, F). This clustering of the AChR receptors, which are induced by the motoneuron protein agrin in vivo, are locations on the myotube where neuromuscular junction formation occurs and another indication of functional myotube formation.
The presence of the ryanodine (RyR) receptor and dihydropyridine (DHPR) receptor clusters, as well as their colocalization in vivo, represents the development of excitation-contraction coupling apparatus in skeletal muscle myotubes [8, 9, 24]. The clustering of both RyR and DHPR receptors was observed on the myotubes after 30 days in culture (Figure 5 A–D). The clustering and colocalization of the RyR + DHPR clusters was observed with a number of different myotube morphologies (Figure 5 E–L). This functional adaptation illustrated that the medium formulation facilitated not only the structural maturation but also the functional maturation of myotubes in this in vitro system. The clustering of the RyR + DHPR receptors was also observed in 90 day old myotubes, indicating that the older myotubes maintained their functional integrity (Figure 6 A–F).
The myotubes contracted spontaneously in the culture and the contractions began generally by day four and continued throughout the life of the culture. Most of the myotubes expressed functional voltage gated sodium, potassium and calcium ion channels as reported previously . The voltage clamp electrophysiology of the myotubes indicated the inward and outward currents that demonstrates functional sodium and potassium channels (Figure 7 A). Figure 7B shows the single action potential fired by the myotubes in current clamp mode. The electrical properties of the myotubes were also consistent with the formation of mature, functional myotubes.
In this report we have documented the development of a system for long-term, functional, skeletal muscle culture. This system was in response for the need to develop more physiologically relevant skeletal muscle myotubes for functional in vitro systems. For our specific research, they are a component needed for the creation of a realistic model of the stretch reflex arc as well as their integration with bio-MEMS cantilevers for screening applications. The results indicate that we have achieved three significant structural modifications within the myotubes, causing both the developmental profile and functionality of the fibers to better mimic in vivo physiology. This skeletal muscle maturation resulted from modifications to the cell culture technique, a new medium formulation and the use of NBactiv4 as the maintenance medium.
We developed this serum-free medium, supplemented with growth factors, that supported the survival, proliferation and fusion of fetal rat myoblasts into contractile myotubes in a semi-empirical fashion. The rational for selecting the growth factors was based on the distribution of their cognate receptors in the developing myotubes in rat fetus [3, 4, 25]. Table 1 and Table 2 reference the literature where these individual growth factors, hormones and neurotransmitters were observed to support muscle and neuromuscular junction development. The composition in Table 1 is the formulation used for a previously published medium used for motoneuron-muscle co-culture and adult spinal cord neuron culture [11, 14]. Table 2 lists the twelve additional factors we have identified in muscle development and neuromuscular junction formation. The use of NBactiv4 for the maintenance of the cells significantly improved the survival of the skeletal muscle derived myotubes despite the original development of NBactiv4 for the long-term maintenance and synaptic connectivity of fetal hippocampal neurons in vitro .
We observed a ratio of 25% neonatal to 75% embryonic MHC expression of the myotubes after 50 days, which contrasts with the previous study in which MHC expression was strictly embryonic. We believe that the myotubes matured in this culture system because the long-term survival provided adequate time for the myotubes to respond to the additional growth factors, which activated the necessary signaling pathways to achieve MHC class switching . This suggests that a different growth factor profile could be utilized to activate alternative signaling pathways and drive myotube differentiation down other pathways. For example, the effects of adding steroid hormones like testosterone to the system could be critically examined.
The colocalization of RyR and DHPR clusters in the myotubes indicated the formation of the excitation-contraction coupling apparatus and was another indication of functional maturation in the fibers. Excitation-contraction coupling is the signaling process in muscle by which membrane depolarization causes a rapid elevation of the cytosolic Ca2+ generating contractile force . The close proximity of the DHPR and RyR complexes occurs at specialized junctions established between the transverse tubule and sarcoplasmic reticulum (SR) membranes in skeletal muscle myotubes . At these junctions, T-tubule depolarization is coupled to Ca2+ release from the SR resulting in muscle contraction [29, 30]. This structural adaptation represents a significant functional change due to the fact that excitation-contraction coupling is required for successful extrafusal muscle fiber development as well as neuromuscular junction formation [8, 9, 23]. This improved model provides the potential to study excitation-contraction coupling in a defined system as well as for myotonic and myasthenic diseases.
The development of sarcomeric structures, the excitation-contraction coupling apparatus and MHC class switching in the skeletal muscle myotubes is a result of the improvements to the model system documented in this research. This improved system supports the goal of creating physiologically relevant tissue engineered muscle constructs and puts within reach the goal of developing functional skeletal muscle grafts. This defined model can also be used to map the developmental pathway regulating the formation of the excitation-contraction coupling apparatus as well as MHC class switching. Furthermore, we believe this serum-free culture system will be a powerful tool in developing advanced strategies for regenerative medicine in muscular dystrophies, stretch reflex arc development and integrating skeletal muscle with bio-hybrid prosthetic devices. Due to the use of a serum-free defined culture system this also has applications for new high-throughput screening systems for use in drug discovery research and toxicology investigations.
The F1.652 monoclonal antibody developed by Helan Blau was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA. This work was supported by NIH grant # 5R01NS050452.