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7 Myogenesis

7.1 Introduction

Myogenesis is the creation of muscle tissue from stem cells in the embryo. Muscle becomes meat, and we are interested in its early origin. Some animals produce high-quality meat, others produce it very rapidly. Can one animal do both?

Bulk meat, such as a steak or roast, is composed of countless microscopic muscle fibres (myofibres). Each myofibre is multinucleate (has numerous nuclei) because the myofibres are very long (usually many centimetres). Thus, one nucleus could not possibly produce enough RNA for protein synthesis in the whole myofibre. How does this multinucleate condition arise?

The components involved are as follows.

Premyoblasts - cells capable of mitosis but not yet producing muscle proteins.
Myoblasts - cells no longer capable of mitosis but now starting to produce muscle proteins.
Myotube - a multinuclear myofibre produced by the fusion of myoblasts.
Secondary fibre - a multinuclear myofibre produced by the fusion of myoblasts on the surfaces of a myotube.
Myofibre - a muscle fibre matured from either a myotube or a secondary fibre.

7.2 Mitosis

Mitosis is cell division. First the nucleus doubles its DNA, then it divides. Then the rest of the cell divides to produce two identical daughter cells. The mesodermal cells of somites and limb buds undergo frequent mitosis, with a variety of factors such as IGF-1 (insulin-like growth factor) and PDGF (platelet-derived growth factor - platelets are cell fragments in the blood stream) being mitogenic (causing mitosis). The peak of mitotic activity in the limb buds of the chick embryo is at about 5 days incubation - we would love to have similar information on cattle, sheep and pigs.

Dividing premyoblasts are rounded in shape (but compressed together) and are locked into a mitotic cycle.

G1, (gap one) or rest after the last mitosis (2.0 hr)
S, synthesis of new DNA (4.3 hr)
G2, (gap two) or rest after DNA synthesis (2.4 hr)
M, mitosis (0.8 hr)
return to G1 or become a myoblast (5-7 hr)

The times given are approximations for premyoblasts growing in the laboratory. They give us a guess of how long these events might take in farm animals. The escape from this cycle - when a premyoblast becomes a postmitotic myoblast - is thought to be irreversible. The cycle preceding a premyoblasts's escape has been termed the quantal division. The number of times a clone of premyoblasts remains locked into the mitotic cycle might have a profound importance on myoblast numbers. Just one extra cycle by all premyoblasts might double the number of myoblasts and give rise to extra myofibres (hyperplasia). The population of premyoblasts capable of mitosis may not be completely homogeneous since it might contain true stem cells and committed precursors. A committed precursor is a cell giving rise to a cohort of 16 terminally differentiated myoblasts. Obviously, factors regulating premyoblast proliferation, such as triiodothyronine (a hormone produced by the thyroid gland and otherwise associated with heat regulation in the body), are extremely important to the meat industry.

Another way of looking at this system of cell proliferation is to consider premyoblasts at the escape point in their mitotic cycle. Both the daughter cells produced by mitosis may stay in the cycle, both may escape to become myoblasts, or one may stay in and one may escape. With a population of cells, the percentage of escaping cells starts at 0% in very young embryos, before the appearance of any myoblasts, and then increases towards, but never reaches 100% (some stem cells remain as satellite cells, a source of muscle nuclei during growth and regeneration).

Cell populations containing mixtures of premyoblast stem cells, mononucleate myoblasts and fused myoblasts can be sorted with arabinocytidine. This prevents the formation of new myoblasts but does allow cell fusion. In cultures from 11-day chick embryos, about 20% of cells are myoblasts, but the percentage is lower in younger embryos. Another way of sorting cells is to determine what percentage may be cloned to give rise to myoblasts capable of fusion. Chick leg bud mesoderm at 72 hours incubation contains 0%, at 80 hours it contains 10%, and at 6 days it reaches 60%. In human limb buds, comparable values are 14% at 36 days, with a 90% plateau from 100 to 172 days.

Another factor controlling cell proliferation might be the duration of the mitotic cycle, possibly by a variation of the duration of G1 . Premyoblasts escaped from the mitotic cycle to become myoblasts eventually fuse together, but the fusion of cells eventually becomes less frequent, as if inhibited. Alternatively, escape from the mitotic cycles may be in late G1. Cells in G1 may respond to PROSTAGLANDIN E1 with a transient increase in intracellular cyclic AMP. This may activate protein kinase and the onset of myoblast fusion.

The nervous system exerts some regulation over muscle development, and its control over myoblast proliferation is probably achieved by varying the duration of G1 rather than G2. Because of the importance of G1 in the regulation of cell numbers, it is interesting to note the G1 -S boundary is the point at which the cell synthesizes calmodulin. Calmodulin is a protein able to bind calcium ions, and is thought to be involved together with cyclic AMP in the regulation of many aspects of cell metabolism, growth and division.

7.3 Myoblasts

The morphological features of premyoblasts are similar to those of other types of precursor cells in the embryo. RNA synthesis dominates cell activity and results in a large oval-shaped nucleus, prominent nucleoli (which vary in number between species), diffuse chromatin (nuclear DNA) and many ribosomes (granules in the cytoplasm responsible for protein synthesis). The large amount of RNA (an acid) in the cytoplasm binds to basic (alkaline) dyes, and the cytoplasm is described as basophilic (base-loving). Myoblasts are bipolar, spindle-shaped cells, whereas fibroblasts tend to be triangular in shape. Myoblasts may form tight junctions where they are in contact with each other, usually at the tips of their elongated cytoplasmic extensions. Here we see myoblasts fusing and becoming lined up.

The process of lining up is very important. It can only occur if the free end of the myoblast at one end of the line attaches itself to an appropriate point at one end of the future muscle and if the free end of the myoblast at the other end of the line attaches itself to the other end of the future muscle. This brings the line of myoblasts into line with the long axis of the muscle. The mechanism may be myoblasts following the lines of connective tissue fibres in the developing muscle (contact guidance). If bad connections are made (say both ends of the line of myoblasts attach to the same end of the muscle) - then the line of myoblasts degenerates. Thus, a developing muscle contains many degenerating myoblasts which have failed to develop appropriate connections. Only if the line of myoblasts is properly attached at both ends can the myoblasts contract, stretch their membranes, and take up amino acids for further protein synthesis.

Fusion is preceeded by a period of cell to cell recognition in which the myoblasts may still be dispersed chemically with EDTA (which removes calcium ions and loosens cell contact). Recognition is followed by a period of adhesion in which trypsin (an ezyme able to attack proteins) must be added experimentally in order to disperse the cells. Finally, after membrane fusion, fused cells cannot be dispersed. Cultured myoblasts fuse when their numbers reach a certain density, perhaps in response to a chemical signal. Within the myoblast, an increase in the level of cyclic AMP initiates the events that lead to fusion. Myoblasts have surface antigens for cell-cell recognition. Myoblast fusion is triggered by calcium ions but is inhibited by magnesium and potassium ions.

7.4 Myotubes

This simple explanation only holds true for muscles with a simple structure - parallel myofibres running from one end of the muscle to the other. Most muscles in meat animals have a complex structure with an angular arrangment of myofibres onto a tendon at one of the muscle. Thus, in most muscles, development occurs in subunits (in bundles of myofibres called fasciculi - the singular is fasciculus).

Within each fasciculus, therefore, lines of myoblasts develop running from one end to the other.

Next, the myotubes start to develop myofibrils. Much more will be said about myofibrils later - here we only need to know they are responsible for muscle contraction and they run longitudinally with their striations in line across the future myofibre. Myofibrils are added around the nuclei and the nuclei remain in the long axis. In the early days of microscopy, the nuclei were difficult to see (because they are only easily visible if they are stained, and appropriate stains had not yet been invented). Thus, early microscopists saw only tubular structures (formed by the myofibrils) and named them myotubes.

7.5 Secondary fibres

Getting myotubes properly lined up in the future muscles takes a long time - and the time for parturition (birth) is rapidly approaching. The muscles have only about 20% of their future myofibres formed by these myotubes. What next? A very rapid process of forming secondary fibres from a new generation of myoblasts.

New myoblasts take advantage of the myotubes being properly lined up in the approriate pattern for the future muscle. The new myoblasts attach themselves to the myotube surface and, when the myotube contracts, it brings the myoblasts into the correct alignment for fusion. The fused myoblasts now become a secondary fibre as they start to produce myofibrils. But the secondary fibre is not yet innervated - it does not contract next time the myotube contracts. So the secondary fibre shears off from the surface of the myotube once it is sufficiently stiffened by its new myofibrils. In this remarkable process of mass production we see 80% of the future myofibres of our meat animals being formed very rapidly just before birth.

7.6 Implications

80% of our meat comes from secondary fibres.
Anything inhibiting myotube contraction will reduce the numbers of future myofibres (for example, loss of amniotic fluid preventing limb movements by the foetus or environmental toxins affecting neuromuscular excitation).
Although both myotubes and secondary fibres go on to become myofibres, they will often become different types of myofibres. Often the myotubes will become slow-contracting myofibres used for fatigue-resistant contraction while secondary fibres will become fast-contracting myofibres used for bursts of strong muscle contraction (which are easily fatigued). Slow-contracting myofibres give meat much of its taste and succulence. Fast-contracting myofibres account for much of the rapid muscle growth in meat animals. Obviously, there are many complexities to add to this simple but true generalization - these will be explained later.
An example of the importance of muscle innervation in meat production!

7.7 Commitment, differentation & maturation

The overall sequence of events in myogenesis may be separated into commitment, differentiation and maturation.

Commitment occurs when a stem celll has its future restricted to myogenesis.
Dfferentiation is marked by the transcription of genes coding for typical features of the myofibre.
Maturation or terminal differentiation occurs after innervation.

Myogenin and MyoD are genes in a family activated when commitment to a myogenic lineage occurs. These genes could be very useful in exploring the factors determining muscle size in meat animals. Myogenin and MyoD are sensitive to thyroid hormones, as well as being regulated by muscle electrical activity, possibly via a mechanism dependent on cyclic-AMP. Innervation controls the abundance of myogenic factors such as MyoD1 and myogenin, and denervated muscle reverts to a neonatal state (that is, cut the nerve to a muscle, and the muscle may revert to the state it had before it was first innervated). Subject to neural regulation, MyoD is prevalent in fast muscles, and myogenin in slow muscles.

Transforming growth factor beta 1 (TGF-ß1) is a small peptide involved the joint develop of myofibres and connective tissues. Following local induction of TGF-ß1, it may produce local gradients enhancing the development of connective tissues by fibroblasts, but inhibiting myogenesis. Thus, a reduction of TGF-ß1 gradients might produce a condition similar to that found in double-muscled cattle.

7.8 Myofibre arrangement

The major nerve trunks grow into a limb bud by following the connective tissue framework of the bud, but developing muscles with more than about 10 myotubes may be necessary to invoke the formation of side branches of nerves to the muscle.
Muscles may be attached to either the shaft (diaphysis) or the knob (epiphysis) of a bone. But the longitudinal growth of bones occurs at cartilagenous epiphyseal plates, and one of these plates is located between each epiphysis and its diaphysis. Thus, to retain their positions relative to each other during epiphyseal plate growth, some muscle attachments must migrate over the bone surface. Muscle migrations are regulated by the bone and traction by the periosteum (the membrane around the bone) is responsible for the migration of tendon insertions.
Muscle development in the limbs of foetal meat animals may be shaped by a dynamic interaction between linear skeletal growth and the resistance of muscles to stretching.
If muscle stretching shapes muscle growth, the determination of myofibre arrangment might be explained by the contact guidance theory attempting to explain how nerve cells invade developing tissues. Myotubes and myoblasts might be guided by a matrix of very fine connective tissue fibres. Migrating myogenic stem cells in chick embryos branch into filopodia at their leading edges, and stem cells follow the alignment of fine connective tissue fibres. The ends of myotubes actively grow through the tissue of the future muscle and have a well developed cytoskeleton dominated by microtubules.
Molecules of fibronectin have binding sites for a number of the components surrounding cells (such as for collagen and glycosaminoglycans) but also they can bind to the surfaces of cells. Thus, matrices of fibronectin may be involved in the guiding of cell migrations and the determination of muscle architecture.
The initial arrangement of connective tissue fibres is probably determined by fibroblasts stretching the extracellular matrix.
Cultured myoblasts only develop a parallel alignment if they are cultured on a type of collagen able to form distinct collagen fibres.
Myotubes may be pulled into alignment by their already anchored ends to follow the dominant directions of a stretched matrix.
The angular arrangement of myofibres is difficult to explain. Perhaps the tensile forces shaping the connective tissue matrix of a pennate muscle are transmitted by intramuscular tendons. Another possibility is myoblast arrangement may be influenced by the orientation of electrical fields. Cultured myoblasts become arranged with their long axes perpendicular to electric fields of 36 to 170 mV/cm.
Intracellularly, the parallel arrangement of myofibrils is dependent on the proper attachment of the whole myotube or secondary fibre. New filamentous proteins for incorporation into myofibrils appear first at the periphery of cells - thus, the longitudinal orientation of filaments may follow the direction of membrane stretching.

7.9 Degeneration and survival

Many of the early histologists who studied myogenesis were impressed by the widespread evidence of cellular degeneration they found in developing muscles. Lysosomes (membranous bags full of deadly digestive enzymes - often called suicide bags) capable of causing degeneration are well developed even in myoblasts. Experimentally, if myofibres are slowly stretched they will continue to develop. But they degenerate if they are not stretched. The passive stretching of myotubes activates the sodium ion pump of their membranes, and this is followed by increases in amino acid uptake and protein synthesis. The stimulation of amino acid transport and protein synthesis induced by the stretching of myotubes may act through the release of messenger substances such as arachidonic acid, diacylglycerol and prostaglandins.

This is a very important point concerning muscle growth - not just in meat animals, but in ourselves as well. We all know exercise encourages muscle development while inactivity allows muscles to waste away. The mechanism involves cell membranes. When myotubes or secondary fibres get properly attached at their ends, they can contract. When they can contract, they can stretch their cell membranes. When their cell membranes are stretched, the uptake of amino acids is enhanced.

Further information

Structure and Development of Meat Animals and Poultry. Chapter 6.