1982; 62:1757-1762.PHYS THER. Neil I SpielholzVivo and In VitroSkeletal Muscle : A Review of Its Development In

http://ptjournal.apta.org/content/62/12/1757services, can be found online at: The online version of this article, along with updated information and

Collections

Anatomy and Physiology: Musculoskeletal System     in the following collection(s): This article, along with others on similar topics, appears

e-Letters

"Responses" in the online version of this article. "Submit a response" in the right-hand menu under

or click onhere To submit an e-Letter on this article, click

E-mail alerts to receive free e-mail alerts hereSign up

by guest on February 21, 2013http://ptjournal.apta.org/Downloaded from

Skeletal Muscle

A Review of Its Development In Vivo and In Vitro

NEIL I. SPIELHOLZ

Physical therapists are well-aware of the form and function of mature skeletal muscle. The steps that muscle goes through to become that way, however, are generally less familiar. This article reviews some highlights of myogenesis as revealed by in vivo and in vitro studies. It also describes how tissue culture may help elucidate the mechanisms of genetic myopathies.

Key Words: Muscle differentiation; Myogenesis; Myopathies, genetic; Tissue cul­ture.

Although skeletal muscle is usually thought of in terms of its muscle fibers, whole muscle is an organ whose function depends on the following four com­ponents: 1. Muscle fibers, the contractile tissue proper, which

constitute the bulk of the muscle because they are so numerous.

2. Connective tissue, which holds the muscle fibers together and harnesses their mechanical activity and also supports the intramuscular vasculature and nerve supply.

3. Blood vessels, which bring in nutrients and take away waste products.

4. Nerves, motor and sensory, for communicating with the CNS. The development of muscle from undifferentiated

embryonic cells has been intensely investigated for many years. The impetus for this work was not solely academic curiosity. Considerable evidence indicates that muscle regeneration, which occurs in many dis­eases and after some types of injury, recapitulates embryonic myogenesis to a great extent.1'2 Knowl­edge gained from further study of normal muscle development may yield clues to understanding and, it is hoped, to treating hereditary myopathies.

HOW MYOGENESIS IS STUDIED

To characterize mature skeletal muscle, one must consider its morphological, biochemical, electrophys­iological, and mechanical properties. Developmental biologists are not only concerned with describing the

evolution of these characteristics as muscle progresses from undifferentiated stem cells to the mature func­tioning muscle. They are interested also in uncovering the mechanisms or control factors that influence and direct the sequential changes that occur.

Two main approaches are used to accomplish these goals. With the first, the in vivo approach, muscle is studied as it develops normally in embryos and after birth. With the second, in vitro techniques are used by which myogenic precursor cells obtained from embryos are grown in tissue culture. Each approach has advantages and disadvantages. Development in vivo proceeds under the control of a multitude of factors, including humoral, neural, and other influ­ences as well as the genetic program of the myogenic cells. Most of these factors are poorly understood and cannot be controlled. In vitro, cells can be cultured using media and conditions that are fairly easy to manipulate; but when grown this way, muscle never differentiates as completely as it does in vivo. Also, some components of the artificial nutrient media (eg, chick embryo extract, horse serum, and fetal calf serum) have not been fully identified. Despite these shortcomings, in vitro techniques have shown the considerable extent to which muscle can develop without the influence of neural or humoral factors, as will be described in this article.

OVERVIEW OF MYOGENESIS

A general overview of myogenesis will be useful before I go into more detail. As outlined in Figure 1, a mature muscle fiber, like any other cell in the body, is the result of a genetic program that begins at fertilization. The striated and multinucleated fiber

Supported in part by a grant from the Muscular Dystrophy Association of America and by Rehabilitation Services Administra­tion 16-P-56801/2.

Volume 62 / Number 12, December 1982 1757

by guest on February 21, 2013http://ptjournal.apta.org/Downloaded from

Fig. 1. Summary of histological events leading to mature muscle fibers. Progeny of undifferentiated mesodermal cells give rise to premyoblasts which after many divisions become myoblasts. These cells, influenced by conditions not yet fully understood, cease dividing and fuse with one another forming myotubes. Myotube nuclei, each one contributed by an individual myoblast, no longer undergo mitosis. Myotubes synthesize contractile proteins and organize them into myofibrils. With further development, myotubes become muscle fibers packed with myofibrils, sarcoplasmic reticulum, T tubules, mitochondria, and other sarcoplasmic organelles.

that physical therapists know so well is actually a syncytium formed during gestation by the fusion of many mononucleated cells called myoblasts. The mul­tinucleated cell thus formed is a myotube. Myotubes then start expressing their muscular identity by syn­thesizing myofibrils, the intracellular organelles of muscle. Obviously, before the myoblasts can fuse, large pools of them are required. Myoblasts are gen­erated by premyoblasts, cells that undergo many di­visions. Some authors use the term myoblast for any myogenic cell, regardless of whether it proliferates or fuses. Other investigators call proliferating myogenic cells premyoblasts and restrict the term myoblast to cells that fuse instead of divide. The former designa­tion is used in this article. Once myoblasts fuse, the cell formed is incapable of further division, that is, its nuclei are postmitotic. Regardless of the terminology used, myogenic precursor cells originate from meso­derm, one of the three embryonic germ cell layers.

With this background, we will now look more closely at muscle as it develops and differentiates in vivo and in vitro.

MYOGENESIS IN VIVO

Early in myogenesis, developing muscle examined under a light microscope does not look like muscle at all. It appears instead as a relatively loose mass of individual mononuclear cells. These cells have a small

amount of cytoplasm surrounding a large nucleus that frequently contains one or more prominent nu­cleoli. Some of these mononuclear cells are myogenic stem cells; others are fibroblasts, whose progeny will form the connective tissue framework of the muscle.

Later in gestation, multinucleated myotubes form in the still-loose meshwork of mononucleated cells (Fig. 2). Although cross-striations are not yet visible with a light microscope, an electron microscope shows that myofibrils are already being synthesized (Fig. 3).

As development progresses, myotubes increase in size and number as more and more myoblasts leave the proliferative cycle and fuse. Myotube diameters also increase as a result of continuing synthesis of myofibrils and other sarcoplasmic constituents found in mature muscle fibers, such as T tubules and sar­coplasmic reticulum.

Even after birth, muscle development is by no means complete. Although the number of fibers a muscle will contain is probably established by this time, fibers continue to hypertrophy (increase in di­ameter) as still more myofibrils are formed (Fig. 4). Fibers also increase in length as body segments elon­gate. Lengthening of muscle is accomplished by the addition of sarcomeres to the ends of fibers, and this continues until growth ceases.3

In addition to postnatal hypertrophy and elonga­tion, subtler changes in muscle also occur after birth. These are related to further differentiation of fibers

Fig. 2. Developing biceps brachii muscle from a 10-day chick embryo (11 days before hatching) seen at the light microscope level. At this age, cross-striated fibers have not yet formed and the tissue looks nothing like adult muscle. Instead, one sees a pale matrix containing two major cell types, namely, light-grey myotubes with large central nuclei (one is bracketed by arrows) and many mononucleated darker cells with small amounts of cyto­plasm (arrowhead points to one). Two larger cells (circled) contain mitotic figures, indicating that a population of cells is still in the proliferative phase. Most nuclei, both in myotubes and in mononuclear cells, possess small, dark nucleoli. (Scale bar is 25 µm.)

1758 PHYSICAL THERAPY

by guest on February 21, 2013http://ptjournal.apta.org/Downloaded from

Fig. 3. Electron micrograph of same muscle as in Figure 2, showing part of a young my-otube. Within the cytoplasm is a central nucleus (CN), some scattered mitochondria (ar­rows), and a few myofibrils in the process of being assem­bled. Clearly seen are dark Z lines bisecting light I bands, and dark A bands with central M lines and lighter H zones. These developing cross-stria-tions were not visible under the light microscope. Outside the myotube (lower right) is a small mononuclear cell (N) with an abundance of small dark ribosomes (arrowhead) in its cytoplasm. In the upper left hand corner, a bit of cyto-plasm(CYT) of another mononuclear cell abuts on the myotube. (Scale bar is 1 urn.)

into slow and fast types. At birth, all mammalian muscle is slow-twitch, based on its isometric contrac­tion times.4, 5 As the animal matures, however, the twitch times of muscles destined to be fast become more rapid; "slow muscles" also speed up at first but then slow again a little bit.4, 5

Twitch times are not the only characteristic that distinguishes adult slow from adult fast muscle. A number of biochemical properties, such as the type of myosin synthesized and adenosinetriphosphatase ac­tivity, are also differentiating factors.6, 7 In fetal mus­cle, however, a correlation linking mechanical and biochemical characteristics is not present. Although fetal muscle is mechanically slow, controversy exists about whether it produces "slow myosin" or "fast myosin." Some investigators have reported that be­fore innervation, developing muscle synthesizes only fast myosin, even though the muscle twitches slowly.8"10 Other studies have found both fast and slow myosin in these muscles.11-13 There is universal agreement, though, that after birth, muscles synthe­size the myosin appropriate for their twitch charac­teristics.

What is the mechanism for postnatal differentiation into final adult slow and fast types? Are the changes a result of a program intrinsic to the maturing fibers themselves, or do other factors, such as innervation, affect this further differentiation? If muscles are de-nervated just before or just after birth, they retain their fetal biochemical characteristics.9 This means they remain either biochemically fast or biochemi­cally mixed, depending on which group of investiga­tors is right. Regardless of this controversy over myo­sin, innervation and subsequent use of muscle have profound effects. Fast muscle speeds up and synthe­sizes only fast myosin. Slow muscle remains essen­tially slow and synthesizes only slow myosin.

MYOGENESIS IN VITRO

Just how far muscle can differentiate in the absence of neural and humoral control is shown by growing muscle in tissue culture. To accomplish this, embry­onic muscle is first dissociated by a combination of mechanical and enzymatic means. This results in a suspension of mononuclear cells; some are myogenic and others are lumped under the classification of fibroblasts.

Fig. 4. Electron micrograph of a biceps brachii muscle from a 3-month-old chicken. Each muscle fiber is now packed with myofibrils, each myofibril showing the char­acteristic components of the sarcomere (SARC). Ele­ments of the internal membrane system of muscle fibers, the sarcoplasmic reticulum and T tubules (arrowheads), are also seen. The dark dots are glycogen deposits. In the chicken, the biceps brachii muscle is composed mostly of white, or type llb fibers. They therefore contain considerable glycogen but relatively few mitochondria. (Scale bar is 1 urn.)

Volume 62 / Number 12, December 1982 1759

by guest on February 21, 2013http://ptjournal.apta.org/Downloaded from

Fig. 5. A. Isolated spindle-shaped cell, presumably a myoblast, 24 hours after starting a culture using embryonic chick pectoralis muscle. Relatively few cells are present in these young cultures. (Scale bar is 25 µm.) B. Same culture four days later. Cells have increased in number as a result of ongoing proliferation. A branched myotube containing many nuclei has also formed. Branched myotubes are frequently seen in muscle cultures but rarely in vivo. (Scale bar is 25 urn.)

Myogenic cells, which are usually bipolar or spin­dle-shaped,14, 15 proliferate for a few days and then fuse to form myotubes (Figs. 5A and 5B). In a few more days, these myotubes become striated (Figs. 6A and 6B). Furthermore, when viewed under phase-contrast optics, which permits one to observe living cells, myotubes in the petri dishes can be seen to twitch sporadically and can be made to contract in unison by electrical stimulation.16 Transmembrane potentials also develop,17 and the myotube's surface becomes sensitive to acetylcholine.18 End-plates, how­ever, do not form in the absence of nerves.

The ability to grow muscle in tissue culture affords unique opportunities for studying different aspects of muscle development. For example, by various manip­ulations of the media nourishing the cells, it has been shown that the metabolizing cells "condition" their media, which in turn influences further development of the cells.19 Conditioning of media is apparently related to cell density. This is an interesting type of

biological feedback. As cells proliferate and reach a particular density, they become concentrated enough to alter their environment. The changed milieu then causes the cells to behave differently—to leave the proliferative cycle and commence fusing.

Another feature of muscle that can be studied in vitro is its intrinsic ability to respond to enforced activity, such as exercise. Because the muscle is living in a petri dish, any response to exercise is independent of neural or humoral influence. This is what is meant by intrinsic ability. Muscle can be exercised in vitro, after myotubes are well-established, by putting sterile stimulating electrodes into the dish and passing small electrical pulses. Muscles react to such stimulation by increasing their rates of myosin synthesis,16, 20 which is presumably related to the hypertrophy that nor­mally accompanies appropriate exercise in vivo. Elec­trical stimulation in vitro has also been shown to affect acetylcholine sensitivity of cultured muscle.21

Fig. 6. A. Striated fiber in a 12-day-old muscle culture flanked by two nonstriated myotubes. The mat of other cells and tissue consists mainly of fibroblasts and extracellular connective tissue fibers. (Scale bar is 25 µm.) B. Electron micrograph of the striated fiber shown in A. Myofibrils are almost fully developed with just some spaces still existing between them. (Scale bar is 1 µm.)

1760 PHYSICAL THERAPY

by guest on February 21, 2013http://ptjournal.apta.org/Downloaded from

SATELLITE CELLS—MYOGENIC STEM CELLS IN ADULT MUSCLE

We have outlined myogenesis in cultures originated from embryonic muscle. Embryos, after all, should afford a plentiful source of undifferentiated myogenic stem cells. But what about older muscle? Can cultures be established, for example, from muscle after birth? A first guess might be that it cannot be done because, as already described, muscle fiber nuclei are postmi­totic. What would be the source of new myogenic stem cells? Experiments have shown, however, that cultures can be established from older muscle, al­though with somewhat more difficulty than from embryonic muscle. Obviously then, myogenic stem cells can be mobilized after birth. But where do they come from?

Briefly, the existence of undifferentiated stem cells in muscle was first described by Mauro, who called them "satellite cells."22 These are mononuclear cells with a small amount of cytoplasm that lie so close to a muscle fiber that when seen with a light microscope they are almost indistinguishable from the more nu­merous subsarcolemmal muscle nuclei. In fact, satel­lite cells are so intimately associated with their muscle fibers that they lie in between the fiber and the fiber's basement membrane, or basal lamina (Fig. 7).

Satellite cells are scattered along the length of a muscle fiber. As muscle develops, satellite cells de­crease in number presumably because some of them become incorporated into the growing muscle fibers.23'24 In adult muscle, probably less than 10 percent of nuclei seen with a light microscope are nuclei of satellite cells.24, 25

Over the years it has become fairly well established that satellite cells are capable of being "activated" under certain conditions in vivo and will then func­tion as premyoblasts. This leads ultimately to the formation of new muscle fibers by the mechanisms already described. Satellite cells, then, are also the source of undifferentiated myogenic stem cells that permit the establishment of cultures from adult mus­cle.

IN VITRO DEVELOPMENT OF GENETIC MYOPATHIES

Because satellite cells are part of the stem cell population from which a muscle develops, muscle afflicted with a genetic primary myopathy contains satellite cells harboring the same genetic defect. Therefore, cultures established from such muscle should ultimately reproduce the disease.26 This will occur in vitro provided the following conditions are met: 1. The defective or mutant gene is indeed being

expressed within the muscle nuclei themselves and not in a different cell, with the muscle being af­fected secondarily.

Fig. 7. Electron micrograph showing the intimate rela­tionship of a satellite cell and its muscle fiber (cross-sectional view). The muscle fiber is indented slightly where the satellite cell fits into it, and the two are sepa­rated by only a thin clear space. Note that the fiber's basement membrane (arrow), also known as the basal lamina or glycocalyx, encloses both the fiber and the satellite cell. Only a thin rim of satellite cell cytoplasm surrounds the satellite cell nucleus. (Scale bar is 1 µm.)

2. Besides the genetic defect, manifestation of the disease does not require extraneous factors oper­ating on muscle, like neural or humoral influences, which are of course absent in tissue culture.

3. The muscle matures or differentiates in vitro to the stage when the genetic defect becomes apparent. For reasons not yet understood, there have been

conflicting reports concerning whether cultures estab­lished from dystrophic muscle behave differently from normal cultures. Other conditions, however, such as adult-onset acid maltase deficiency26 and some other less-common myopathies27 have been re­capitulated in vitro. Hopefully, as work continues along these lines, future findings will be translated into helping to alleviate human neuromuscular dis­eases.

SUMMARY

Although muscle is usually thought of in terms of its ability to develop tension, this specialized function culminates an intricate developmental sequence. As more is learned concerning the dynamics of normal myogenesis, including the roles played by neural and humoral influences and the effects of enforced activity or inactivity, better methods for treating patients with muscular disorders are likely to follow.

Acknowledgments. The author is grateful for the expert technical assistance of Michael Gorycki and Michael Pugliese in culturing and preparing tissue for the microscopy shown.

Volume 62 / Number 12, December 1982 1761

by guest on February 21, 2013http://ptjournal.apta.org/Downloaded from

REFERENCES

1. Allbrook D: Muscle regeneration. Physiotherapy 59(8):240-247, 1973

2. Allbrook D: Skeletal muscle regeneration. Muscle Nerve 4:234-245, 1981

3. Williams PE, Goldspink G: Longitudinal growth of striated muscle. J Cell Sci 9:751-767, 1971

4. Buller AJ, Eccles JC, Eccles R: Differentiation of fast and slow muscles in the cat hind limb. J Physiol (Lond) 150:399-416, 1960

5. Close R: Dynamic properties of fast and slow skeletal mus­cles of the rat during development. J Physiol (Lond) 173:74-95,1964

6. Barany M: ATPase activity of myosin correlated with speed of muscle shortening. J Gen Physiol 50:197-218, 1967

7. Kugelberg E: Adaptive transformation of rat soleus motor units during growth: Histochemistry and contraction speed. J Neurol Sci 27:269-289, 1976

8. Chi JCH, Rubinstein N, Strahs K, et al: Synthesis of myosin heavy and light chains in muscle cultures. J Cell Biol 67:523-537, 1975

9. Rubinstein N, Kelly AM: Myogenic and neurogenic contribu­tions to the development of fast and slow twitch muscles in rat. Dev Biol 62:473-485, 1978

10. Rubinstein N, Holtzer H: Fast and slow muscles in tissue culture synthesize only fast myosin. Nature 280:323-325, 1979

11. Masaki T, Yoshizaki C: Differentiation of myosin in chick embryos. J Biochem (Tokyo) 76:123-131, 1974

12. Gauthier GF, Lowey S, Hobbs AW: Fast and slow myosin in developing muscle fibres. Nature 274:25-29, 1978

13. Schiaffino S, Askanas V, Engel WK, et al: Myosin isoenzymes in cultured human muscle. Arch Neurol 39:347-349, 1982

14. Konigsberg IR: Clonal analysis of myogenesis. Science 140:1273-1284, 1963

15. Konigsberg IR: The embryological origin of muscle. Sci Am 211(2):61-66, 1964

16. Brevet A, Pinto E, Peacock J, et al: Myosin synthesis in­creased by electrical stimulation of skeletal muscle cell cul­tures. Science 193:1152-1154, 1976

17. Rash JE, Fambrough D: Ultrastructural and electrophysiolog­ical correlates of cell coupling and cytoplasmic fusion during myogenesis in vitro. Dev Biol 30:166-186, 1973

18. Hartzell HC, Fambrough D: Acetylcholine receptor produc­tion and incorporation into membranes of developing muscle fibers. Dev Biol 30:153-165, 1973

19. Konigsberg IR: The role of the environment in the control of myogenesis in vitro. In Rowland LP (ed): Pathogenesis of Human Muscular Dystrophies. Amsterdam, The Netherlands, Excerpta Medica, 1977, pp 179-198

20. Stockdale FE, Brevet A, Pinto E, et al: Myosin synthesis in electrically stimulated cultures of skeletal muscle cells. In Rowland LP (ed): Pathogenesis of Human Muscular Dystro­phies. Amsterdam, The Netherlands, Excerpta Medica, 1977, pp 826-834

21. Cohen SA, Fischbach GD: Regulation of muscle acetylcho­line sensitivity by muscle activity in cell culture. Science 181:76-78, 1973

22. Mauro A: Satellite cell of skeletal muscle fibers. Journal of Biophysical and Biochemical Cytology 9:493-495, 1961

23. Moss FP, Leblond CP: Satellite cells as a source of nuclei in muscles of growing rats. Anat Rec 170:421-436, 1971

24. Schultz E: A quantitative study of the satellite cell population in postnatal mouse lumbrical muscle. Anat Rec 180:589-596, 1974

25. Wakayama Y: Electron microscopic study on the satellite cell in the muscle of Duchenne muscular dystrophy. J Neuro-pathol Exp Neurol 35:532-540, 1976

26. Askanas V, Engel WK, DiMauro S, et al: Adult-onset acid maltase deficiency: Morphologic and biochemical abnormal­ities reproduced in cultured muscle. N Engl J Med 294:573-578, 1976

27. Askanas V: Regeneration of diseased muscle in vitro as a tool to study the pathogenesis of human neuromuscular disorders. In Mauro A, et al (eds): Muscle Regeneration. New York, NY, Raven Press, 1979, pp 297-304

1762 PHYSICAL THERAPY

by guest on February 21, 2013http://ptjournal.apta.org/Downloaded from

1982; 62:1757-1762.PHYS THER. Neil I SpielholzVivo and In VitroSkeletal Muscle : A Review of Its Development In

Information Subscription http://ptjournal.apta.org/subscriptions/

Permissions and Reprints http://ptjournal.apta.org/site/misc/terms.xhtml

Information for Authors http://ptjournal.apta.org/site/misc/ifora.xhtml

by guest on February 21, 2013http://ptjournal.apta.org/Downloaded from