These mice have demonstrated that NFATC2 regulates development and differentiation of several tissue types. Wild-type mice controls for each of the mutant mice were generated from heterozygous matings. Male mice between 8 and 12 wk of age were used for these studies. All animals were handled in accordance with the institutional guidelines of Emory University. Tibialis anterior TA , soleus, and masseter muscles were collected using standardized dissection methods, embedded in TBS tissue freezing medium Fisher Scientific , and frozen in isopentane cooled in liquid nitrogen.
For the soleus, the CSA of sections throughout the soleus muscles was measured using computer-assisted morphometric measurements, and the region spanning the belly of the soleus was subsequently used for all further analyses described below. For the masseter and TA, anatomical markers of each muscle were used to find the same region in different samples, and these sections were subsequently used for analysis. TA muscles were subject to a standardized local freeze damage as described previously Pavlath et al.
In experiments analyzing regenerating TA muscles, sections containing the largest area of damage were selected for measurements. The core of the damaged area, as defined by the region that was the least regenerated, was visualized, and the image was captured to a computer screen. In addition, the CSA of individual regenerating myofibers was also measured at various time points after damage. To analyze particular fiber types, cross sections of soleus muscles were stained with antibodies against type 1 BA-D5 Schiaffino et al.
After washing in TNT 0. Nuclei within the dystrophin-positive sarcolemma were counted for — myofibers, and the number of myonuclei was expressed per myofibers. After 2 h, cells were placed in DM and allowed to differentiate for 48—72 h before fixation in 3. Myotubes were grouped into two categories, those with two to four nuclei and those with five or more nuclei.
The percentage of myotubes in each category was calculated. Production of infectious retrovirus and infection of primary myoblasts were performed as described previously Abbott et al. Myoblasts in well plates were infected by two rounds of infection with a combination of either control or NFATC2 retroviruses and an NFAT-responsive reporter retrovirus Abbott et al.
Cells were collected and assayed for luciferase as described previously Abbott et al. Immunoblots were performed as described Friday et al. After high stringency washing, membranes were visualized by autoradiography. All RT reactions were performed using 2. Our previous studies in cultured muscle cells indicated that individual NFAT isoforms are specifically induced to undergo nuclear translocation in response to changes in intracellular calcium at different stages in myogenesis Abbott et al.
As seen in the top of Fig. This size difference correlates with a decrease in the CSA of individual myofibers Fig. A small decrease in the percentage of myofibers expressing type I MyHC occurs, but no difference exists in the percentage of type II myofibers right.
The CSA of an entire muscle is affected not only by the size of individual myofibers but also by the number of myofibers. This decrease in myofiber number is a result from a defect in myofiber formation during embryonic development. Since calcineurin-dependent signaling pathways have been proposed to regulate MyHC isoform expression Chin et al.
These muscles were chosen for analysis because they differ from the soleus in several properties. In addition, the masseter exhibits differences in myoblast populations compared with limb muscles Pavlath et al.
A similar decrease is observed in the masseter Fig. During postnatal development and during regeneration after injury, myofibers grow in size, a process involving addition of nuclei to the myofiber. Since myonuclei are postmitotic, myofiber growth involves the fusion of muscle precursor cells with myofibers.
These muscle precursor cells, called satellite cells, lie underneath the basal lamina surrounding each myofiber and are in close juxtaposition to the myofiber itself. Satellite cells are normally quiescent but become activated and start proliferating in response to growth factors. Since satellite cells are required for skeletal muscle regeneration Robertson et al.
At this time point, satellite cells have become activated and are proliferating and beginning to differentiate. We next analyzed the ability of satellite cells to fuse and form new myofibers. When myofibers form after injury, their nuclei are centrally localized, facilitating identification of regenerating myofibers. The number of centrally nucleated myofibers in the core of the injury 7 d after injury was counted as described in Materials and Methods. At this time point, the area of injury is completely filled with these regenerating myofibers.
As seen in Fig. Therefore, satellite cell function appears normal in the early stages of muscle regeneration in the absence of NFATC2. C A comparison of hematoxylin and eosin—stained sections of regenerating TA muscles at day 25 after injury demonstrates the smaller size of the mutant myofibers top.
The CSA of regenerated myofibers in the central region of the lesion was determined for various time points after injury bottom. To determine if NFATC2 regulates further growth of regenerating myofibers, the CSA of regenerating myofibers was measured at different times after injury.
As further growth of the regenerating myofibers occurs, the CSA of myofibers in the mutant at each time point is significantly decreased compared with wild-type Fig. Thus, the function of NFATC2 is required for growth of myofibers as suggested by our previous in vitro studies Abbott et al. Myofiber growth is dependent on both nonmuscle and muscle cells. A small amount of myogenin is detected in both types of myoblasts, presumably due to spontaneous differentiation in the cultures.
A portion of a Coomassie-stained gel demonstrates relative protein loading. C The number of nuclei within individual myotubes at least two nuclei was counted. Myotubes were grouped into two categories, and the percentage of myotubes in each category was determined. Since myofiber size correlates with myonuclear number Allen et al. To clearly define the nuclei within myotubes, the myotubes were immunostained for EMyHC and the number of nuclei within individual myotubes at least two nuclei was counted.
The percentage of the myotubes in each category was calculated. In addition, an increased number of myotubes occurs in the mutant cultures data not shown.
To confirm that the absence of NFATC2 is responsible for the observed defects in myotube size and nuclear number in vitro, two types of experiments were performed. First, the expression levels of other NFAT isoforms in the mutant cells were analyzed to rule out compensatory changes that could contribute to defects in myotube size. NFATC4 was not detected in either genotype data not shown. These results are consistent with the results of Ranger et al.
NFATC4 is not expressed in myoblasts of either genotype data not shown. Cells were induced to differentiate, and luciferase assays were subsequently performed. Data are reported as fold increase in luciferase activity over control cells. D The number of nuclei within individual myotubes at least two nuclei was counted for each of the retrovirally infected cultures.
Myotubes were grouped into two categories as in the legend to Fig. Myoblasts infected with either control or NFATC2 retroviruses were induced to differentiate, and luciferase activity was determined. No significant difference is observed in nuclear number of wild-type cells infected with either control or NFATC2 retrovirus data not shown , suggesting that overexpressing NFATC2 in wild-type muscle cells does not affect size or nuclear number of myotubes.
Since fusion of myoblasts and thus addition of myonuclei is required for growth of mammalian myofibers Darr and Schultz ; Rosenblatt and Parry ; Mozdziak et al. A A representative wild-type myofiber immunostained with an antibody against dystrophin red and stained with DAPI blue illustrates the myonuclear number assay. Arrow indicates a myonucleus within the dystrophin border, and arrowheads indicate nuclei outside the myofiber.
We have shown previously that individual NFAT proteins translocate to the nucleus of muscle cells at specific stages of myogenesis Abbott et al. To determine if this defect is due to impaired myofiber growth, we examined regenerating skeletal muscle as a model for myofiber growth. Defects in myofiber growth are intrinsic to muscle cells since cultured muscle cells lacking NFATC2 form small myotubes with few nuclei. Muscle growth results from an ordered sequence of events.
Withdrawal of mitogens in vitro causes myoblasts to exit the cell cycle and activate the expression of differentiation specific genes. Subsequently, myoblasts migrate toward one another, elongate, align, and through cell—cell interactions fuse together to form a multinucleated cell. Multiple proteins are required for migration and fusion of myoblasts such as integrins and other cell adhesion molecules, metalloproteases, and phospholipases Knudsen In addition, increases in intracellular calcium Constantin et al.
Once myotubes form, additional myoblasts fuse with the myotube, and the myotube grows in size. Postnatal muscle growth in vivo is also characterized by myoblast fusion with myofibers, leading to an increase in myonuclear number. Numerous studies indicate that myoblasts are critical for muscle growth in vivo. When the proliferative capacity of myoblasts is attenuated, increases in myonuclear number and myofiber size are blocked in growing rats Darr and Schultz ; Rosenblatt and Parry ; Mozdziak et al.
Additionally, the lack of myoblast growth factors such as leukemia inhibitory factor Kurek et al. Thus, numerous molecules contribute to proper muscle cell size. The decrease in the number of nuclei is correlated with a decrease in myotube size, implicating an NFATC2-dependent pathway in the control of myotube size.
Thus, the downstream targets of NFATC2 allow the fusion of differentiated muscle cells with newly formed myotubes and the subsequent growth of the myotube. Based on our results, we present the model outlined in Fig.
Increases in intracellular calcium lead to the activation of calcineurin. NFATC2 may either directly or indirectly regulate gene transcription of a cell surface protein pathway 1 that mediates cell—cell interaction or cell fusion between mononucleated muscle cells and newly formed myotubes. Evidence exists for cell surface proteins that mediate the fusion of myoblasts with myotubes.
The integrin very late antigen 4 on multinucleated muscle cells and its counterreceptor vascular cell adhesion molecule 1 on myoblasts are thought to mediate myoblast fusion with myofibers during development Rosen et al.
In addition, glycoproteins may mediate myoblast—myotube interactions to allow fusion and muscle growth, since wheat germ agglutinin can block myoblast fusion with myotubes and decrease myotube size Muroya et al. Alternatively, NFATC2 could either directly or indirectly regulate gene transcription of a secreted protein pathway 2 that recruits differentiated mononucleated myoblasts to fuse with adjacent myotubes.
Secreted factors apparently can regulate the fusion of cells with myotubes. Fibroblasts cocultured with young or old myotubes can acquire myogenic characteristics and fuse with myotubes Breton et al. Though not shown, both pathway 1 and 2 could also mediate myotube—myotube fusion. Specific molecules downstream from NFATC2 that contribute to muscle growth, though not examined in this study, are currently being investigated. Only myoblast fusion is represented in the model. NFATC2 may regulate a cell surface protein pathway 1 that mediates cell—cell interaction or cell fusion between differentiated muscle cells and newly formed myofibers.
Alternatively, NFATC2 could regulate gene transcription of a secreted protein pathway 2 that recruits differentiated muscle cells to fuse with adjacent myofibers. Myotube—myotube fusion has been suggested to occur during regeneration Robertson et al.
Several lines of evidence implicate multinucleated cells as controlling the site and extent of myoblast fusion. During the development of mammalian skeletal muscle, primary myofibers form initially and are followed by the formation of secondary myofibers. Primary myofibers control the site of secondary myofiber assembly, since secondary myofibers form only at the site of innervation on the primary myofiber, independently of the nerve Duxson et al.
In addition, the primary myofiber seems to restrict the fusion of secondary myoblasts, whereas the secondary myofiber seems to recruit fusion, since secondary myoblasts fuse primarily with the forming secondary myofiber Harris et al. This specificity of fusion shares analogy with Drosophila muscle development in which founder myoblasts express dumbfounded, an attractant for myoblast fusion, and recruit fusion-competent myoblasts to fuse with founder myoblasts and not with other myoblasts Ruiz-Gomez et al.
Further suggesting that myofibers can control the location of myoblast fusion, myofibers elongate by fusion of myoblasts at their ends during mammalian postnatal growth Williams and Goldspink ; Zhang and McLennan The mechanisms by which myofibers regulate the fusion of myoblasts are unknown but may involve expression of proteins such as those mentioned above. This control of fusion by multinucleated muscle cells is likely one mechanism by which the size of muscle cells is regulated.
Calcineurin has been shown to be involved in skeletal muscle hypertrophy Musaro et al. The inability of NFATC2 overexpression to induce an increase in myotube size and nuclear number in wild-type myotubes suggests that NFATC2 is not involved in skeletal muscle hypertrophy.
Several possibilities exist to explain this lack of effect on growth of wild-type myotubes. This hypothesis is supported by the fact that NFATC2 cannot be activated by a calcium ionophore in mature myotubes Abbott et al. These pathways could involve the activation of additional transcription factors necessary for forming a transcriptional complex with NFATC2.
Thus, wild-type myotubes already may be at their maximal cell size and no further increases are possible even in the presence of the recombinant NFATC2.
In summary, we demonstrate that the calcium-regulated transcription factor NFATC2 regulates muscle growth. These data implicate an NFAT-dependent pathway in newly formed myotubes that controls further cell fusion in a mechanism that is distinct from the initial formation of multinucleated muscle cells. Myoblast fusion with multinucleated cells is central to proper muscle development but is also clinically relevant.
Enhancing myoblast fusion with multinucleated cells has potential therapeutic value by improving the fusion of endogenous myoblasts in the treatment of muscle injuries or muscle atrophy or by enhancing the fusion of exogenous transplanted myoblasts in gene therapy protocols Blau and Springer Each cell is spindle shaped with a single nucleus and no visible striations Figure 4.
Watch this video to learn more about muscle tissue. In looking through a microscope how could you distinguish skeletal muscle tissue from smooth muscle? The three types of muscle cells are skeletal, cardiac, and smooth. Their morphologies match their specific functions in the body. Skeletal muscle is voluntary and responds to conscious stimuli. The cells are striated and multinucleated appearing as long, unbranched cylinders. Cardiac muscle is involuntary and found only in the heart. Each cell is striated with a single nucleus and they attach to one another to form long fibers.
Cells are attached to one another at intercalated disks. The cells are interconnected physically and electrochemically to act as a syncytium. Cardiac muscle cells contract autonomously and involuntarily. Smooth muscle is involuntary. Each cell is a spindle-shaped fiber and contains a single nucleus.
No striations are evident because the actin and myosin filaments do not align in the cytoplasm. You are watching cells in a dish spontaneously contract. They are all contracting at different rates, some fast, some slow. After a while, several cells link up and they begin contracting in synchrony. Discuss what is going on and what type of cells you are looking at. The cells in the dish are cardiomyocytes, cardiac muscle cells. They have an intrinsic ability to contract.
When they link up, they form intercalating discs that allow the cells to communicate with each other and begin contracting in synchrony. Under the light microscope, cells appear striated due to the arrangement of the contractile proteins actin and myosin. Skip to content Learning Objectives Describe the characteristics of muscle tissue and how these dictate muscle function.
By the end of this section, you will be able to: Identify the three types of muscle tissue Compare and contrast the functions of each muscle tissue type. External Website. Chapter Review The three types of muscle cells are skeletal, cardiac, and smooth.
Interactive Link Questions Watch this video to learn more about muscle tissue. Skeletal muscle cells are striated. Review Questions. Critical Thinking Questions You are watching cells in a dish spontaneously contract. Why does skeletal muscle look striated?
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