Structure of Myofibers

Structural and physiologic features of skeletal muscle determine much of its response to injury. Although muscle cells are frequently called muscle fibers or myofibers, they are in fact multinucleated cells of considerable length, which in some animals may approach 1 m. Myonuclei are located peripherally in the cylindrical myofiber (Fig. 15-1) and direct the physiologic processes of the cellular constituents in their area through a process known as nuclear domains. This anatomic arrangement allows segments of the cell to react independently of other portions of the cell. Myonuclei are considered terminally differentiated, with little or no capacity for mitosis and thus for regeneration.

Associated with myofibers are the satellite cells, also known as resting myoblasts (Web Fig. 15-1). These cells are distributed along the length of the myofiber, between the plasma membrane (sarcolemma) and the basal lamina. Satellite cells in skeletal muscle are very different from cells of the same name found within the peripheral nervous system. Muscle satellite cells are fully capable of dividing, fusing, and reforming mature myofibers. Thus, under favorable conditions, muscle cells (myofibers) are able to fully restore themselves after damage. Recent studies have found that pluripotent cells derived from bone marrow can also contribute to skeletal muscle repair, albeit only to a very small degree.

Each myofiber is surrounded by a basal lamina and outside of this by the endomysium, a thin layer of connective tissue containing capillaries. Myofibers are organized into fascicles surrounded by the perimysium, a slightly more robust layer of connective tissue (Web Fig. 15-2). Entire muscles are encased in the epimysium, a protective fascia that merges with the muscle tendon. This connective tissue framework is not inert, but in fact forms an integral part of the contractile function of muscle by storing and relaying force generated by myofiber contraction.

Ultrastructural examination reveals that skeletal muscle is a highly and rigidly organized tissue, with what are perhaps the most highly structured cells in the body. Each myofiber is composed of many closely packed myofibrils containing actin and myosin filaments. The striations visible with light microscopy (Fig. 15-2) represent the sarcomeric arrangement of muscle cells, in which actin and myosin filaments attached to transverse Z bands form the framework, and other organelles and intracytoplasmic materials are interspersed within this framework (Fig. 15-3). The endoplasmic reticulum of myofibers is called the sarcoplasmic reticulum and is modified to contain terminal cisternae that sequester the calcium ions necessary to initiate actin and myosin interaction and thus contraction. Sarcolemmal invaginations that traverse the cell, the T (for transverse) tubules, allow rapid dispersion of a sarcolemmal action potential to all portions of the myofiber. The terminal cisternae of two adjacent sarcomeres and the T tubule form what is called the triad (Fig. 15-3, A).

Neuromuscular junctions can only be visualized using electron microscopy or other specialized procedures (Fig. 15-4). Neuromuscular junctions occur only in specific zones within the muscle, usually forming an irregular circumferential “band” midway between myofiber origin and insertion.

Types of Myofibers

Mammalian muscles are composed of muscle fibers of different contractile properties. A common classification of these fibers is based on three major physiologic features: (1) rates of contraction (fast or slow), (2) rates of fatigue (fast or slow), and (3) types of metabolism (oxidative, glycolytic, or mixed). These physiologic differences form the basis of histochemical methods that demonstrate fiber types. There are several fiber-type classifications. Classification of fibers into type 1, type 2A, and type 2B (Table 15-1) has proved to have practical application in muscle pathology. It is the classification used in this text. Type 1 fibers are rich in mitochondria, rely heavily on oxidative metabolism, and are slow-contracting and slow-fatiguing. Type 2 fibers have fewer mitochondria and are glycolytic, fast-contracting, and more easily fatigable. In most species, type 2 fibers can be subdivided into type 2A and type 2B. Type 2B fibers are the fast-contracting, fast-fatiguing, glycolytic fibers that depend on glycogen for their energy supply. Type 2A fibers are mixed oxidative-glycolytic and therefore, although fast-contracting, are also slow-fatiguing. Thus type 2A fibers are “intermediate” in the concentration of mitochondria, fat, and glycogen between type 1 and type 2B.

Most muscles contain both type 1 and type 2 fibers, and these can be demonstrated by the myosin adenosine triphosphatase (ATPase) reaction (Fig. 15-5, A). Notice that the different fiber types are normally intermingled, forming what is called a mosaic pattern of fiber types. In most mature muscles, the staining pattern of the ATPase reaction reverses when sections are preincubated in an acid rather than an alkaline solution. There are examples of both patterns in the illustrations in this section. Acid preincubation can also be used to distinguish type 2A and type 2B fibers (Fig. 15-5, B). Regenerating fibers, classified as type 2C fibers, stain darkly in both acid and alkaline preparations, which is a distinguishing feature. In most species, oxidative enzyme reactions to demonstrate mitochondria also demonstrate fiber types to some degree (Fig. 15-6, A). Fiber typing can also be done by utilizing immunohistochemical procedures to identify specific myosin isoforms.

The percentage of each fiber type varies from muscle to muscle (Fig. 15-7). Type 1 fibers (slow-contracting, slow-fatiguing, and oxidative) are plentiful in those muscles in which the main function is slow, prolonged activity, such as those that maintain posture. Type 1 predominant postural muscles are most often located deep in the limb. Within the same muscle, the percentage of type 1 fibers often increases in the deeper portions. Muscles that contract quickly and for short periods of time, such as those designed for sprinting, contain more type 2B fibers. Only rarely are muscles composed of only one fiber type (e.g., the ovine vastus intermedius is type 1). Athletic training causes some type 2B fibers to be converted to 2A. There are also variations within breeds and differences in the same muscle in different species. For example, the dog has no type 2B purely glycolytic fibers; all canine fibers have strong oxidative capacity (see Fig. 15-6, B).

Innervation and Motor Units

The axons of the peripheral nerve trunks contain terminal branches that innervate multiple myofibers. The terminal branches form synapses with the myofibers at the neuromuscular junction. The myofibers innervated by a single axon form a motor unit, all fibers of which will contract simultaneously after stimulation. Different muscles have different sized motor units that relate to their function. For example, extraocular muscle function does not call for forceful contraction, but rather for many fine movements to smoothly move the globe. Therefore these muscles have very small motor units, with only a small number of myofibers (1 to 4) innervated by each axon. In contrast, the quadriceps muscle is not designed for fine movement, but instead is designed for generation of force, therefore motor units are quite large, with many myofibers (100 to 150 or more) innervated by a single axon.

Gross Pathology and Muscle Sampling

Gross examination includes evaluation of changes in size (atrophied, hypertrophied, or normal), color, and texture. The gross pathologic appearance of skeletal muscle can be quite deceiving. What appear to be mild changes in muscle on gross examination often can be severe on microscopic examination, and what appear to be severe changes on gross examination can turn out to be artefact. Subjective evaluation of size can be highly unreliable unless control muscles (e.g., from normal animals or from the opposite sides) are available for weighing and measuring.

Color changes are common. The intensity of the red color of muscle varies, depending on the type of muscle, the age and species of animal, and the extent of blood perfusion. Pale muscle can indicate necrosis (Fig. 15-9, A and B; see Figs. 15-26; 15-34, A; 15-36, A; and 15-40) or denervation (Fig. 15-9, C; see Fig. 15-37) but is also common in young animals and anemic animals. Pale streaking of muscle most often reflects myofiber necrosis and mineralization (see Fig. 15-9, A and B) or infiltration by collagen or fat (see Fig. 15-9, C and D), and is one of the more reliable indicators of gross pathologic changes. Muscle parasites can be grossly visible as discrete, round to oval, pale and slightly firm zones (see Figs. 15-41 and 15-42, A). Dark red mottling of skeletal muscle can indicate congestion, hemorrhage, hemorrhagic necrosis (see Figs. 15-32, A, and 15-38), inflammation, or myoglobin staining after massive muscle damage (see Fig. 15-36, A) or can simply reflect vascular stasis (hypostatic congestion) after death. Hemorrhagic streaks within the diaphragm often accompany death caused by acute exsanguination. A green discoloration can indicate either eosinophilic inflammation (Fig. 15-10) or severe putrefaction. Lipofuscin accumulation in old animals, especially cattle, can cause a tan-brown discoloration of muscle. Black discoloration of the fascia occurs in calves with melanosis as an incidental finding and in older gray horses with metastasis of dermal melanoma to muscle fascia.

Evaluation of texture is also important. Severely thickened and often calcified fascia occurs in cats with fibrodysplasia ossificans progressiva. Fat infiltration or necrosis can result in abnormally soft muscle. Decreased or increased muscle tone can be caused by denervation. Decreased tone can also occur as a result of a lack of muscle conditioning or postmortem autolysis.

Careful microscopic examination of multiple muscles is often required to detect lesions. In cases of suspected neuromuscular disease, multiple muscle samples should include active muscle (tongue, diaphragm, intercostals, and masticatory muscles), proximal muscle (lateral triceps, biceps femoris, semimembranosus, semitendinosus, and gluteal), and distal muscle (extensor carpi radialis, cranial tibial). For purposes of a biopsy, certain muscles (e.g., lateral triceps, biceps femoris, cranial tibial, semimembranosus, and semitendinosus) are easier to sample because of their parallel myofiber orientation. The ideal samples will also vary, depending on the suspected disorder, such as a type 1 predominant postural muscle for diagnosis of equine motor neuron disease; a type 2 predominant locomotory muscle for diagnosis of equine polysaccharide storage myopathy (EPSSM); and temporal or masseter muscle for diagnosis of masticatory myositis in dogs and masseter myopathy in horses. Short fibers, such as those in the intercostal muscle, are preferred for physiologic studies in which intact muscle fibers are necessary and for studies of neuromuscular junction zones.

To ensure proper fixation and orientation of sections prepared from fixed specimens, the sample should be a strip of muscle no more than 1 cm in diameter, with myofibers running lengthwise. Muscle maintains the ability to contract for some time after death, with the time varying, depending on the physiologic state.

Contraction of muscle after contact with fixative is the most common cause of an artefact called contraction band artefact. Contraction can be prevented or at least minimized by use of a specially designed muscle clamp (Web Fig. 15-3, A) or by placing the sample on a rigid surface, such as a portion of a tongue depressor, and fixing the ends with sutures, staples, or clamps before submersion in the fixative (Web Fig. 15-3, B).

Microscopic Examination

Frequently, lesions in muscles can be detected and evaluated only by microscopic examination. Proper microscopic examination requires evaluation of both transverse and longitudinal sections. Myofiber diameters, cytoarchitectural changes, and the percentage of abnormal myofibers are most reliably evaluated in transverse sections. Longitudinal sections reveal the length of changes such as segmental necrosis or regeneration or deposition of storage material. Improperly oriented samples, which result in sections that have obliquely oriented myofibers and thus neither longitudinal nor transverse myofibers, are difficult to evaluate. Use of a magnifying glass or dissecting microscope can aid in determining the orientation of myofibers during trimming of muscle before sectioning. Routine stains, such as hematoxylin and eosin (H&E), run the risk of offering the pathologist a “vast pink wasteland” for evaluation (Fig. 15-11, A) and are often inadequate for detecting subtle myopathic changes, lesions within intramuscular nerves, or presence of abnormal stored material. Various special stains, including reticulin, Masson trichrome, von Kossa, lipid (performed on frozen sections of fixed samples), and periodic acid–Schiff (PAS) for glycogen, are often invaluable in evaluation of routinely processed skeletal muscle (Web Table 15-1). Examples of many of these disorders can be found here in this chapter. Other valuable stains and reactions can only be performed on frozen sections of unfixed muscle samples (see Web Table 15-1).

For many decades, myofiber typing could be done only on frozen sections using the myosin ATPase reaction. Recently, immunohistochemical staining of myosin has been developed for demonstration of myofiber types in formalin-fixed muscle. This is a major advantage because fiber-type staining is often essential for the complete evaluation of muscle. It is most useful in demonstrating preferential involvement of a fiber type and alteration of the fiber-type pattern, the result of denervation and reinnervation.

Other Methods of Evaluation

Physiologic testing of isolated intact myofibers in vitro forms the basis for diagnosis of malignant hyperthermia (MH). Short fibers, such as from samples of intercostal muscle, are preferred. While maintained in a physiologic solution, myofiber bundles are exposed to various agents, such as caffeine and halothane, to detect abnormal contractural sensitivity. Biochemical and molecular biologic analysis of muscle samples can evaluate levels of muscle enzymes and other proteins, and genetic analysis can be performed to detect specific gene defects. These latter tests require fresh muscle samples snap-frozen in liquid nitrogen and maintained at −70° C until analysis.