Excitation

Excitation begins with the origination of a nerve impulse within the central nervous system. The impulse exits the spinal cord, travels along a motor neuron, and arrives at the motor end plate of an individual myofiber. Motor end plates are located in deep primary clefts or folds of the sarcolemma. The nerve impulse arrives at the motor end plate, releasing acetylcholine, which traverses the synaptic cleft and binds to nicotinic receptors. The binding of acetylcholine produces a conformational change in the nicotinic receptor, which results in the opening of ion channels, producing increased sodium and potassium conductance in the end plate membrane and causing depolarization and generation of an end plate action potential. The T tubule system propagates the action potential across the sarcolemma in all directions and carries it deep into the muscle fiber. Diffusion of acetylcholine from the receptors or hydrolysis by acetylcholine esterase causes depolarization to cease.

Excitation-contraction coupling

Coupling of excitation with contraction begins with the arrival of the action potential at the triad region, which causes opening of sodium channels embedded in the T tubule membrane and rapid influx of sodium into the cell. The rapid influx through the sodium channel results in a change of polarity across the membrane that we associate with depolarization. As a result the inside of the sarcolemma becomes more positive relative to the outside of the membrane. Depolarization of the T tubule membrane triggers a voltage-gated calcium channel known as the dihydropyridine receptor, which is also located within the T tubule membrane. Activation of the dihydropyridine receptor triggers the closely related ryanodine receptor in the terminal cisternae of the sarcoplasmic reticulum, causing it to release calcium into the sarcoplasm.

Contraction

Calcium released into the sarcoplasm binds to troponin C, resulting in a conformational change of the tropomyosin molecule and unmasking part of the actin filament responsible for binding to myosin: the G-actin monomer. With binding of myosin to the G-actin monomers, a 90-degree cross-bridge forms. As soon as the cross-bridge forms, myosin ATPase cleaves ATP to form adenosine diphosphate (ADP) and adenosine monophosphate (AMP), resulting in another conformational change converting the 90-degree angle to a 45-degree angle. The contractural force is generated by the movement of the cross-bridge myosin head from a 90-degree to a 45-degree angle with a coincident sliding of the myofilaments relative to one another toward the center of the sarcomere. The hydrolytic products of ATP then detach from the myosin head enabling the cycle to recommence. The addition of new ATP to the myosin molecule results in the rapid dissociation of the actin and myosin filaments.^4,5 Sustained contraction occurs with the rapid repetition of this mechanicochemical cycle.⁵

Each sarcomere shortens in unison in successive ratchetlike movements composed of multiple acts of relaxation and reengagement depending on the degree of shortening demanded by the nervous system. With input from the central nervous system to relax opposing muscles, the process of excitation-contraction coupling produces smooth coordinated movement.

Relaxation

For the cell to relax and return to baseline polarity, the sarcoplasmic reticulum and the plasmalemma must reaccumulate calcium, thereby lowering the concentration of calcium in the sarcoplasm and the T tubule while sarcolemmal membranes begin to repolarize. Sarcoplasmic calcium concentrations decrease when calcium-magnesium ATPase pumps return calcium to the sarcoplasmic reticulum and sodium-potassium ATPase pumps move sodium extracellularly and potassium intracellularly. Energy-dependent reaccumulation of calcium by the sarcoplasmic reticulum results in the release of calcium from troponin C, restoration of the resting configuration of the troponin-tropomyosin complexes, and ultimately disruption of the linkage between the myosin and actin filaments. Further details on muscle structure and contraction are available.^4–7

An important note is that all of the processes necessary for relaxation are active, meaning they require energy in the form of ATP for them to occur. In the absence of ATP, actin and myosin remain engaged, and the muscle remains in the shortened or contracted position known as rigor.

Several terms are frequently used when referring to the shortening of muscle in veterinary medicine. Contraction implies a shortening or tightening of the muscle that is initiated by the central nervous system. This electrical activity is recorded readily using electromyography. A contracture is considered to be the shortening of a muscle because of abnormal activity within the muscle cell or cells. A contracture originates within the muscle fiber itself and is therefore electrically silent on electromyography. Contractures occur in disorders characterized by muscle cramping such as polysaccharide storage myopathy or recurrent exertional rhabdomyolysis in which muscle shortening is typically electrically silent. The term spasm is more generic and may refer to a transient or sustained contraction or contracture. If a spasm is painful, the term cramp is often used. Fibrous tissue within a muscle may prevent complete stretching and shortening of a muscle, thereby limiting the range of motion of a limb or joint. This process occurs in fibrotic myopathy as described in a subsequent section.

Source of Energy for Contraction

Understanding the sources of energy for contraction is important because most metabolic myopathies arise from derangements in these processes. ATP stores in muscle are very minimal and are quickly exhausted. Thereafter, ATP must be generated to sustain muscle function. In short, ATP is generated by either aerobic (oxidative) or anaerobic mechanisms. The primary substrates for aerobic metabolism include circulating nonesterified fatty acids and glucose or intramuscular stores (or both) of glycogen and triglycerides.

When the phosphate bond of ATP is cleaved, the resulting by-products include ADP, inorganic phosphate, and energy. At the onset of muscle activity, cleavage of the phosphate bonds of creatine phosphate (or phosphorylcreatine) in the presence of ADP by the enzyme creatine kinase produces creatine and ATP. This anaerobic pathway is quickly supplanted by other pathways to produce energy (Table 11-1).

TABLE 11-1 Energy Sources in Muscle^∗

Numerous different processes within the muscle fiber generate ATP after creatine phosphate and include glycogenolysis, glycolysis, Krebs cycle, β-oxidation of free fatty acids, and purine nucleotide deamination. Which pathways are most active depends on factors that include the underlying muscle fiber composition, the number of motor units recruited, capillarization of the muscle, the underlying oxidative and glycolytic capacities of the muscle fibers, and the delivery of oxygen and other energy substrates to the muscle. In turn, the breed and age of the horse, the length or stage of exercise, the intensity of exercise, and the fitness level of the horse influence these factors.

For specifics on glycolysis and generation of ATP via the Embden-Meyerhof pathway or the hexose monophosphate shunt, the reader is directed to a physiology text. Glycolysis or the breakdown of glucose (a 6-carbon molecule) to carbon dioxide and water in the presence of oxygen liberates 38 moles of ATP per mole of glucose. Whereas the fatty acid oxidation of a similar 6 carbon fatty acid yields 44 moles of ATP.

The primary sources of glucose include circulating glucose in the blood, small quantities of free glucose within the sarcoplasm, and stored glycogen within the muscle cell. Glycogen is a branched polymer of individual glucose molecules consisting of 1:4α and 1:6α linkages. Breakdown of glycogen, or glycogenolysis, results in the liberation of 37 moles of ATP per mole of glycogen. A large, extramuscular storage site of glycogen is the liver. With exercise, the liver begins to liberate free glucose into the blood. Muscle cells take up glucose by glucose transporters located in the cell membrane. Athletes who have exercised frequently over time develop a denser network of capillaries about each muscle cell to facilitate the delivery of oxygen, free fatty acids, and glucose to the cell and the removal of by-products.

Glycogen is made through the action of glycogen synthase and glycogen branching enzyme. The breakdown of glycogen’s 1:4α linkages is mediated by phosphorylase, whereas the breakdown at the 1:6α linkages is mediated by glycogen debranching enzyme. To prevent a futile cycle of formation and degradation, these sets of enzymes are reciprocally regulated. Glycogen synthase is stimulated when glucose-6-phosphate is plentiful, whereas the presence of glucose-6-phosphate and ATP inhibit phosphorylase. Phosphorylase is activated when levels of AMP are elevated. Hormones may also activate phosphorylase. Epinephrine stimulates phosphorylase in both the liver and skeletal muscle, leading to elevations in blood lactate and glucose. Glucagon acts only on liver phosphorylase, resulting in elevations in blood glucose and not lactate.

Glycolysis and glycogenolysis are rapid processes and produce adequate energy for aerobic exercise. When exercise intensity increases and oxygen demand cannot be met, glucose is metabolized anaerobically. Anaerobic glycolysis of glycogen yields 3 moles of ATP and lactic acid as a by-product. The accumulation of lactic acid within the cell lowers the pH of the sarcoplasm, inhibits the function of many intracellular enzymes, and contributes to fatigue.

After a period of light aerobic exercise (this varies between individuals but in many is after approximately 15 to 20 minutes), the muscle begins to rely more on the β-oxidation of free fatty acids than on glycolysis for the production of ATP. Small stores of fat are located within the muscle. However, most of the free fatty acids are liberated from body fat stores or the liver and are taken up by the muscle cell during exercise. The longer the aerobic exercise, the more energy is derived from metabolism of free fatty acids. More intensive aerobic exercise uses a combination of carbohydrate and fatty acids as energy sources. The amount of energy derived from fatty acid metabolism appears also to be partially influenced by diet. The higher the intake of fat, the more muscle appears to rely on free fatty acids for energy. The optimal ratio of fat in the diet of the horse has yet to be determined.

The onset of fatigue during aerobic exercise is different from that during anaerobic exercise. During aerobic exercise, glycogen depletion, hyperthermia, and electrolyte depletion appear to be major factors, whereas during anaerobic exercise, lactic acidosis and depletion of creatine phosphate and ATP appear to be important factors initiating fatigue. Accumulation of lactic acid within the muscle cell leads to decreased intracellular pH and inhibition of the enzymes important in glycolysis. A low intracellular pH also inhibits excitation-contraction coupling. In aerobic and anaerobic forms of exercise, eventual onset of myalgia and decreased motivation appear to be additional factors in fatigue.

When most forms of energy within the muscle are exhausted the last source of energy within the cell is the formation of ATP from two molecules of ADP in a process known as the myokinase reaction. A by-product of this reaction is ammonia. Increasing ammonia concentrations in the circulation are well correlated with depletion of ATP. Depleted fibers may develop a painful, electrically silent contracture that is similar to rigor, and severely affected fibers may die. The death of muscle cells related to exercise is known as exertional rhabdomyolysis. Rhabdomyolysis is often assessed by the measurement of enzymes such as creatine kinase or lactate dehydrogenase that are released into the blood when muscle cells are severely stressed (the cell membranes become permeable) or the cells lyse.

Differentiation

Under light microscopy using simple staining techniques, skeletal muscle appears to be a homogeneous tissue. However, muscle is composed of many fibers with differing characteristics. Myofibers may be grouped into two categories: type 1 and type 2. Table 11-2 gives a summary of the characteristics of different fiber types. The type 1 and 2 classification is broad, and a spectrum of fibers has characteristics of both categories. The differences between fibers arise from variability in the proteins that comprise the contractile elements. Several different isoforms have been identified for the myosin heavy and light chains, tropomyosin, troponin I, troponin T, and troponin C. The greatest variability is in the isoform of the myosin heavy chain. In addition, the calcium pumping capacity of the sarcoplasmic reticulum may vary from cell to cell.

TABLE 11-2 Fiber Type Characteristics

All types of fibers are found in most skeletal muscles. Type 1 fibers are high in oxidative capacity and therefore rely on glucose, free fatty acids, and a high amount of oxygen to supply energy for contraction. Their sarcoplasmic reticula have a moderately slow reuptake of calcium capacity. Therefore type 1 fibers are best suited to moderate contraction speeds and sustained contractions over long periods. As expected, these fibers predominate in muscles involved in posture. Type 2 fibers have a fast myosin ATPase rate and a rapid capacity to reuptake calcium into the sarcoplasmic reticulum. They are large-diameter fibers compared with type 1 fibers and have a higher glycolytic capacity, making them suited to powerful fast contractions. These fibers predominate in the skeletal muscles involved in locomotion and rapid fine-motor movements such as around the eye. Type 2 fibers that have activities closer to type 1 fibers are called type 2A, whereas those that exhibit high anaerobic capacity are characterized as type 2B. Because of the high oxidative capacity of type 1 fibers, these muscles often appear to have a deeper red color than fibers high in type 2A and 2B fibers.

Histologically, differentiation of muscle fiber types relies on the measurement of myofibrillar ATPase activity (the enzyme responsible for the breakdown of ATP at the actin-myosin cross-bridges). Staining for this enzyme distinguishes two distinct fiber types at pH 9.4. The slow-twitch type 1 fibers have a low activity at this pH and so appear lighter in color than the fast-twitch type 2 fibers. The type 2 fibers can be divided further by preincubation at a more acidic pH into types 2A, 2B, and even 2C (the pH values required for this differentiation vary with the laboratory but are around 4.5 and 4.3).

The varying capacities of muscle fiber types is largely the result of variations in myosin heavy-chain isoforms. These isoforms are identified through antibody and electrophoresis techniques. In mammals, at least nine have been identified. Two are developmental (embryonic and neonatal), two are found in cardiac muscle (alpha and beta), three are common in skeletal muscle (2a, 2b, and 2x), and two are rare with greatest expression in jaw, extraocular, and laryngeal muscles. Beta is also found in skeletal muscle and is an isoform with properties of slow twitch, high oxidative capacity, low glycolytic capacity, and low ATPase activity similar to the histologically identified type 1 myofibers. Type 2a myosin heavy-chain isoforms are typically found in fibers exhibiting fast twitch, high glycolytic, high ATPase activity, and high oxidative capacity and as such resist fatigue. Type 2b and 2x isoforms are found in muscles that are fast twitch, produce strong contractions, have high glycolytic and ATPase activity but have low oxidative capacity and, as such, fatigue faster than type 2a rich myofibers.^8–11

Innervation

A neuron and all of its innervated myofibers constitute a motor unit. Each motor neuron may innervate a few or several hundred myofibers, and all of those myofibers exhibit similar contraction characteristics. Each myofiber, however, is innervated only by a branch of one motor neuron. Two types of motor nerve fiber are found, slow and fast. The type and pattern of electrical activity on a muscle cell is related to the type of myosin isoforms expressed: The large phasic motor nerve fibers innervate the type 2 myofibers, whereas the small tonic fibers innervate the type 1 fibers.¹¹

Muscle spindles are a bundle of specialized muscle fibers that run parallel to nonsensory muscle fibers and which contact sensory axon endings. Muscle spindle cells are important in telling the nervous system about the tone and length of the muscle. They are also important in maintaining posture. Discharges are relayed directly to the ipsilateral spinal motor neurons that innervate the muscle in which the spindle is located, resulting in a reflex arc. Two types of sensory neurons innervate each spindle: the primary endings are from the large-diameter group 1A afferent axons, whereas the less elaborate secondary endings are from the smaller group 2 afferent axons. The spindle cell fibers also receive motor neuron innervation from small γ-motor neurons, which helps keep the spindle taut when the muscle contracts, which, in turn, enables the sensory endings to respond to a wide range of muscle lengths.

Muscle spindle cells are different from the muscle Golgi tendon organ. Golgi tendon organs are found at the ends of the muscle fibers at the musculotendinous junction and are formed from a common tendon to which several muscle fibers are attached. A sensory nerve ending wraps around each Golgi tendon organ. The sensory nerve innervating the Golgi tendon organ responds to tension generated by the contraction of any of these muscle fibers, and the discharge is relayed to the ipsilateral α-motor neurons, where it is inhibitory. This action forms another reflex arc responding to muscle tension and helps ensure that muscles of flexion and extension do not contract simultaneously.

Fiber Recruitment

Under neural control, an orderly selection of fibers occurs with increasing demands. When just maintaining posture or walking the horse uses only the nerves supplying the slow-twitch type 1 and possibly a few of the intermediate type fast-twitch high-oxidative type 2A fibers. As the speed or intensity of work increases, more and more fibers are recruited. Thus, at a medium trot, approximately 50% of the muscle cells within a muscle belly are contracting, whereas at a gallop, most or all fibers are involved. The fibers are recruited in a set order: type 1, type 2A, and then type 2B. The gradation in response of a muscle is known as the recruitment of motor units. A motor unit consists of a motor nerve and the cells it innervates. The smoothness of a muscular contraction occurs because of the asynchronous contraction of the various motor units.

Distribution

The proportions of fiber types present within a muscle vary according to individual traits of the horse, the muscle location, the breed, and the age of the horse.^{1,8,10,12–15} In some muscles (in particular the middle gluteal muscle, which is sampled most commonly) the distribution also depends on the sampling site or depth because the distribution of fiber types is nonhomogeneous.^1,10,15,16 The suggestion has been made, however, that within a specific muscle of an individual, the variation in fiber types is small if samples are taken from the same site or an identical contralateral site under controlled conditions.^15,17 This arrangement is why it is generally recommended to biopsy specific muscles to diagnose certain disorders. It provides a relatively homogeneous baseline from which to evaluate a muscle. The exception is when a specific muscle is affected (e.g., exhibits atrophy) wherein it should be biopsied.

General Response of Muscle to Injury

Muscle responds to injury or damage in a limited number of ways. A growing body of evidence indicates that myopathic conditions share a similar final pathway of muscle fiber degeneration, although the number and type of fibers affected and the degree of damage varies.²⁷ The final common pathway involves failure to sequester calcium ions from the sarcoplasm. Prolonged calcium accumulation within the sarcoplasm leads to activation of cellular enzymes and prolonged activation of myofilaments. This process is visible histologically as hypercontraction. Consequently, mitochondria take up excess calcium in an attempt to prevent cellular damage. Accumulation of calcium within the mitochondria leads to failure of energy production (i.e., ATP) within the cell and eventually failure of all intracellular energy-dependent mechanisms, which leads to failure of membrane pumps and ultimately cell swelling and lysis.^27,28

The exact nature of these calcium-activated degenerative pathways is still unknown. A key step may be calcium-induced membrane phospholipid hydrolysis via the activation of phospholipase enzymes, resulting in the production of tissue-damaging metabolites. Other processes, however, involving nonenzymatic lipid peroxidation also may be activated. During exercise the flow of oxygen increases. At the same time an overall depletion of ATP sources occurs. The resultant metabolic stress leads to a greatly increased rate of oxygen free radical production that may exceed the scavenger and antioxidant defense systems of the cell, leading to a loss of cell viability and damage. The increase in free radical activity can lead to a failure of calcium homeostasis and consequent muscle damage, or, alternatively, calcium overloading may lead to an activation of free radical–mediated processes.²⁹ Multiple initiators likely lead to this final common pathway, depending on the type of exercise, fitness level, presence or absence of underlying genetic defects in muscle metabolism, and nutritional status of the horse. Free radical–induced skeletal muscle damage may be especially important if reperfusion follows a period of ischemia.³⁰

Atrophy and Hypertrophy

Atrophy may be defined as a decrease in muscle fiber diameter or cross-sectional area. Atrophy can occur in a variety of circumstances, including denervation, disuse, and cachexia, in many muscle diseases, after circulatory disturbances, and after extensive myolysis. Within 2 or 3 weeks after peripheral denervation, up to two thirds of the original muscle mass may be lost, although this may not always be obvious clinically because of the presence of superficial and intramuscular fat deposits. Histologic changes reflect the affected nerve and the muscle fiber supplied. In disuse (e.g., atrophy resulting from a tenotomy) a preferential atrophy of type 1 fibers is apparent, often with hypertrophy of type 2 fibers. In contrast, with cachexia and malnutrition, type 2 fibers are preferentially affected, especially in the postural muscles. Not all muscles show the same degree of atrophy, even within one disease process. In cachexia, the back and thigh muscles tend to be the first affected, and the loss of muscle is usually symmetric, with a concurrent loss of fat deposits. A localized asymmetric atrophy is associated with paralysis, immobilization, and denervation.³¹

As indicated previously, hypertrophy may occur through training. A compensatory hypertrophy may also occur in the fibers surrounding an area where fibers have been lost or have decreased greatly in size. Often, large fibers are visible histologically in such conditions, with evidence of incomplete longitudinal division.³¹

Repair after Denervation

Denervation results in atrophy, as remarked previously, but also leads to abnormal muscle excitability. Muscle becomes more sensitive to circulating acetylcholine. The result is the presence of fine, irregular contractions or fibrillations detectable using electromyography. Fibrillations are not visible clinically and should not be confused with fasciculations that appear grossly as jerky muscle contractions in response to pathologic spinal motor neuron discharges.¹¹ With reinnervation the fibrillations cease. Reinnervation can occur in two ways. Damage without severing the nerve is called axonotmesis. The axon is damaged but the Schwann cell and endoneural fibrous sheaths remain. Axons from the proximal part of the damaged nerve may reestablish connections within the empty residual Schwann cell sheath to the distal portion of the original nerve. Initially, atrophy of scattered muscle fibers is visible, but after reinnervation a proportion of the muscle fibers are restored to their normal size and function. Even if the affected muscle is not fully restored, other surrounding unaffected muscles may be able to compensate so that overall function is maintained, although slight gait abnormalities may remain.

The second type of reinnervation occurs when some nerve fascicles are severed completely or when the nerve lesion is located a great distance from the muscle, for example, when lesions involve the motor neuron cell bodies or nerve roots or both. In this case, collateral reinnervation from adjacent, unaffected axons can occur. Given that the nerve type characterizes the muscle type, this action results in regrouping of muscle fibers so that clusters of fibers of the same histochemical type are found rather than the normal checkerboard pattern. This phenomenon is known as fiber type grouping.

Repair after Injury

Regeneration where a break in the continuity of the muscle fiber has occurred differs considerably from neurologic damage. In such cases, repair involves multiplication of nuclei and formation of new internal structures and organelles followed by fusion and alignment into a new multinuclear myofiber. Regeneration can be swift when parts of the affected fiber remain intact. In this case, regeneration occurs at the healthy end of the severed fiber (continuous or budding regeneration). When damage is more severe, regeneration occurs by fusion of mononuclear myoblasts to form a myotube, which develops in a similar way to fetal fibers (discontinuous or embryonic regeneration). The origin of myoblasts is primarily satellite cells, and their origins and function have been well reviewed.^32,33 If the scaffolding, basement membrane, and supporting tissues remain intact, and if the initiating disease process subsides, new fibers tend to orient in a way similar to the original fibers. This type of regeneration usually occurs with segmental necrosis in which necrosis of the whole diameter of the fiber has occurred involving several sarcomeres. Fibroblastic and vascular reactions are minimal in this type of regeneration. Therefore full function of the muscle is restored without residual dysfunction.

Massive trauma, hemorrhage, infection, or infarction can result in damage to the basement membrane and other supporting structures resulting in complete disorientation of the regenerating fibers with significant proliferation of fibroblasts and vessels. In such cases, regeneration may result in significant deformation of the muscle with disruption of its normal function. Regenerated cells often have centrally located nuclei and larger diameters than older myofibers.

Plasma and Serum Enzyme Activities

The enzymes that are most useful in evaluating the equine muscular system are creatine kinase (CK), aspartate transferase (AST), and lactate dehydrogenase (LDH). A change in the plasma activity of any enzyme can occur for a variety of reasons, including alteration in the permeability of the enclosing cell membrane, cell necrosis, impaired removal or clearance of the enzyme, and increased or impaired synthesis. Decreases in plasma enzyme activities are not usually clinically significant. Often, no one specific organ of elimination is responsible, although most elimination occurs via the liver, kidneys, and lungs. Therefore, under most circumstances, the elimination rate of an enzyme from the plasma remains fairly constant, and the rate of influx to the plasma is the crucial factor.

Increases most commonly occur because of a defect in the integrity of the membrane containing the enzyme-rich sarcoplasm.^6,35 The defect results from complete disruption of the cell or a transient change in the permeability of the membrane without cell death. A complete explanation for the pattern of release of intracellular constituents from diseased muscles cannot be given here. Most of the enzymes that are detectable in increased concentrations in the blood with the various muscle disorders are the major soluble (sarcoplasmic) enzymes, although one can also detect the mitochondrial form of AST with severe injury.

The degree of muscle damage does not correlate with the magnitude of increase in serum muscle enzyme activity; a small amount of severely damaged muscle may release a similar amount of enzyme as a larger quantity of mildly damaged muscle. Under general circumstances, the rate of efflux of an enzyme most likely depends not only on its molecular weight and intracellular localization but also on its binding to various intracellular structures and relative intracellular and extracellular concentration. No explanation for the significant differences in half-lives of the various plasma muscle enzymes reported for human beings and horses has been given.

Creatine Kinase

Creatine kinase is the enzyme responsible for breaking down creatine phosphate to creatine and phosphate, releasing energy for muscular contraction. This reaction is the sole source of energy in muscle at the initiation of exercise. Thereafter, energy is supplied by oxidation of glucose and free fatty acids as described previously. Because this enzyme is responsible for the breakdown of creatine phosphate, it is often called creatine phosphokinase. This term, however, is inappropriate, and one should use creatine kinase. In the horse, CK is found mainly in skeletal muscle, the myocardium, and the brain.³⁶ Little or no exchange of CK between the cerebrospinal fluid and plasma appears to occur. A significant increase in total plasma CK activity therefore is caused by cardiac or skeletal muscle damage. CK (molecular weight, 80,000 daltons [Da]) does not enter the bloodstream directly after its release from the muscle cell but transits through the lymph via the interstitial fluid. The total quantity of circulating CK in the horse under normal conditions is estimated to be equivalent to the quantity of CK in approximately 1 g of muscle; a threefold to fivefold increase in plasma CK activity corresponds to the apparent myolysis of approximately 20 g of muscle.³⁷ However, as described previously, making assumptions as to the amount of muscle damaged based on serum CK activity is difficult because muscle cells appear to be able to release significant quantities of CK without necessarily being lysed.

In human beings, two monomers of CK are known and are designated M and B. The enzyme is dimeric, and three possible primary forms exist: MM, MB, and BB. In simplified terms, MM is found mainly in skeletal muscle, BB in the brain and epithelial tissues, and MB in the myocardium. In the horse, some confusion over CK isoenzymes exists, with workers reporting different electrophoretic bands and tissue activities, perhaps because of the different techniques used.^38–41 In one study the heart and skeletal muscle were found to contain predominantly the MM dimer; the brain, pancreas, and kidney, mainly the BB dimer; and the intestine, the MB and BB dimers.³⁹ This work suggests that in the horse, CK isoenzymes on their own could not be used to differentiate between skeletal and cardiac muscle damage. This problem is alleviated largely by the current availability of tests for cardiac troponin I. Unfortunately, commercial tests for skeletal troponin I are not available. In the absence of clinical cardiac disease, elevations in CK primarily can be attributed to skeletal muscle. The plasma half-life of CK in the horse is short (108 minutes, 123 ± 28 min with a plasma clearance of 0.36 ± 0.1 ml/kg/min)^37,42 in contrast to reports of 12 hours in human beings.⁴³

Aspartate Aminotransferase

AST is found mainly in skeletal muscle, liver, and heart, although lower activities are present in several other tissues. Therefore AST is not tissue specific.^36,44,45 Two isoenzymes have been identified by electrophoresis: MAST (found exclusively in the mitochondria) and CAST (originating from the cytoplasm or sarcoplasm). The ratio of cytosolic to mitochondrial enzyme in horse serum is significantly greater than that found in human beings and many other mammals.⁴⁶ In the horse, the ratio of these two forms varies between tissues, and no tissue specificity is apparent for either isoenzyme. The plasma half-life of AST in the horse is 7 to 10 days,⁴² far longer than the 11.8 hours in human beings.⁴³

Lactate Dehydrogenase

Lactate dehydrogenase is a tetrapeptide that occurs in five different isozymic forms produced by combinations of two kinds of subunits (H [heart] and M [muscle]) in groups of four to produce the functional tetrameric molecule. The five isoenzymes are labeled as LDH₁ to LDH₅, or H₄, MH₃, M₂H₂, M₃H, and M₄, respectively. Similar to AST, LDH is found in most tissues and is therefore not organ specific. However, tissues contain various amounts of the LDH isoenzymes, and the isoenzyme profile obtained by electrophoretic separation has been used to identify specific tissue damage.⁴⁷ For the most part, LDH₅ (plus some LDH₄) is found in the locomotor muscles, the liver contains mainly LDH₃ (with some LDH₄ and LDH₅), the heart contains mainly LDH₁ (with LDH₂ and LDH₃), and all types have been found in certain nonlocomotor muscles.^38,41 Training has been shown to increase the percentage of LDH₁ to LDH₄ and decrease that of LDH₅ in skeletal muscle.⁴⁸ The practitioner should use nonhemolyzed samples for LDH determinations because red blood cells contain large amounts of LDH. In general, though, assays for CK and AST are most commonly used to evaluate skeletal muscle.

Urinalysis: Myoglobinuria

Myoglobin (molecular weight 16,500 Da) is essential for the transport of oxygen into and within muscle cells. Most mammalian muscles contain approximately 1 mg myoglobin per gram of fresh tissue, and the suggestion has been made that acute destruction of at least 200 g of muscle must occur before serum myoglobin levels rise sufficiently for detection in the urine.⁴⁹ Serum myoglobin has a short half-life, and therefore measuring serum myoglobin is of limited diagnostic value. In human beings, myoglobinuria occurs in a variety of conditions, including myocardial infarction, crush and burn injuries, malignant hyperthermia, idiopathic and exertional rhabdomyolysis, and certain genetic metabolic abnormalities.^6,49–52 In the horse, myoglobinuria has been seen in equine exercise or trauma-induced rhabdomyolysis,^53,54 white muscle disease in foals,⁵⁵ and postanesthetic myositis.⁵⁶

Pigmenturia or dark urine may be caused by increased myoglobin, hemoglobin, or whole blood in the urine. Low levels of myoglobin, hemoglobin, or whole blood may occur in the urine and can be detected by urine dipstick or laboratory test before visible changes in the color of urine. Therefore considerable amounts of myoglobin, hemoglobin, or whole blood must be present for visible pigmenturia to occur. Many causes of hemolysis in the horse can result in hemoglobinuria, including oxidative damage to erythrocytes,⁵⁷ neonatal isoerythrolysis, hepatic disease, and renal disease.⁵⁹

Unfortunately, stored urine, concentrated urine, and urine containing myoglobin, hemoglobin, or other porphyrins can appear similar in color^57,59,60 (Figure 11-2). Therefore myoglobinuria cannot be distinguished by color alone. The orthotoluidine-impregnated strips commonly used by veterinary clinicians (urine dipstick tests such as BM-Test-8, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Conn.) are insensitive to the presence of myoglobin and hemoglobin.^54,61 The differential salting out of hemoglobin with ammonium sulfate gives false results in human beings^49,50,59,62 and the horse but is used commonly in many laboratories.^{49,50,59,61,62} Visual inspection of the serum or plasma may indicate the cause of the pigmenturia. The low affinity of myoglobin for haptoglobin means that myoglobin is excreted at plasma concentrations around 0.2 g/L, whereas hemoglobin only appears in the urine at plasma concentrations greater than 1.0 g/L. At this concentration a pink discoloration of the plasma occurs, indicating that hemoglobin is present.^9,49,50,63

FIGURE 11-2 Four urine samples. A, With 3 mg/ml hemoglobin. B, With 3 mg/ml myoglobin. C, Normal stored for 1 month at 18° C. D, Control (fresh urine).

On electrophoretic separation on cellulose acetate, myoglobin migrates as a β₂-globulin and hemoglobin as an α₂-globulin. This method can distinguish the two proteins in urine containing high concentrations (>125 μg/ml) of either protein, provided no other proteins, apart from albumin, are present. Immunoassays are the most sensitive and specific tests and can be used for detecting small amounts of myoglobin in blood and urine.^61,64,65 Sequelae of acute tubular necrosis and acute renal failure are associated with myoglobinuria in human beings⁶⁶ and horses.^44,63 Renal disease associated with myoglobinemia in the horse seems to be associated most with horses that are highly stressed (causing decreased renal blood flow), dehydrated, or have lactic acidosis associated with rhabdomyolysis. Therefore horses suffering from “capture” myopathy (e.g., cast in a stall or trailer accident and struggling for a prolonged period) and endurance horses are most at risk.

Muscle Biopsy

The practitioner can obtain muscle biopsy samples by surgical excision under general or local anesthesia⁶³ or by percutaneous needle biopsy from the standing horse.^9,14,67 Because of the variation in fiber composition in muscles, which muscle is chosen, as well as the position and depth of sampling, is important. Good specimen preparation for frozen sections is vital to prevent artifacts such as ice crystals.^6,9,68 For laboratories that prefer examination of frozen sections, specimens are typically shipped chilled overnight so that the laboratory may freeze the sample to minimize artifacts. Other laboratories prefer formalin-fixed specimens. The practitioner should discuss sample procurement, preparation, and shipment procedures with the laboratory before biopsy.

Muscle biopsy allows the morphologic, biochemical, and physiologic properties of the myofibers to be examined with the animal still alive and causes little morbidity. Typical sites used for percutaneous biopsy with a 6-mm modified Bergstrom needle in cases of generalized muscle disease or for research purposes include the semimembranosus, the biceps femoris, and most commonly the middle gluteal muscle. Although convenient, the necessity to have a modified Bergstrom needle means that most practitioners opt for open biopsy of the semimembranosus muscle over percutaneous middle gluteal muscle biopsy. Instructions on how to obtain a muscle biopsy are available online.⁶⁹

To biopsy the semimembranosus a 6- to 10-cm² area is clipped and surgically prepared slightly lateral to and below the anus. At this location, if a scar occurs after the procedure the tail will hide it. An inverted L lidocaine block of the skin and subcutaneous tissues is performed. Local anesthetic is not injected into the muscle itself, which may induce artifact into the biopsy. The muscle belly is innervated poorly for pain, and reaction to transection of a small piece of muscle is minimal. A piece of muscle measuring approximately 1 × 1 × 2 cm is dissected free, and sutures are used to close the subcutaneous layer and skin. Buried skin sutures that do not need to be removed or skin staples appear to be preferable because they are less likely to cause skin irritation and therefore are less likely to be rubbed out by the patient. Should the horse open the biopsy site, the wound typically heals well by second intention with general wound care. This procedure rarely has complications; however, the horse should be current on tetanus prophylaxis as a general precaution. The biopsy is wrapped loosely in saline-dampened gauze and placed in a sealed container for shipment on icepacks to a laboratory skilled in muscle biopsy assessment.

Histochemical staining of frozen muscle sections aids in the detection of glycolytic enzyme deficiencies and storage of various atypical metabolic compounds.^6,70–72 Pathologic changes that can be found in muscle include presence of degenerate or regenerate myofibers, abnormal cytoplasmic inclusions, accumulations of polysaccharide, changes in fiber volume, number, shape, malformation, degeneration or proliferation of organelles, and disruption of the basic architecture of the fiber. Aggregates of inflammatory cells, reactive changes in vessel walls, occlusion of blood vessels, and increased amounts of fibrous tissue are other possible pathologic findings. In some conditions, such as tetanus or botulism, no significant changes may be seen. Further information on the types of changes that occur is available.^6,31

Various histologic stains have been developed to highlight the different morphologic changes and inclusions that occur. The most important of these is the periodic acid–Schiff stain that highlights glycogen stores within a cell and membrane structures containing mucopolysaccharide, glycoproteins, mucoproteins, glycolipids, or phospholipids. Large aggregates of abnormal polysaccharide occur in Quarter Horses and Draft-breed horses with polysaccharide storage myopathy. Histochemical staining for fiber type enables the pathologic changes to be recognized as affecting all fibers or those of one type only. In cases of reinnervation, large groups of a single fiber type are visible as opposed to the more interspersed pattern typically observed.

Immunocytochemical studies have been used to investigate neuromuscular disorders in human beings and to diagnose autoimmune streptococcal-associated myositis in the horse.^73,74 Specialized staining techniques help to localize specific enzymes and intracellular and extracellular muscle components such as the various collagen and myosin types, complement, and fibronectin. Electron microscopic studies enable investigation into the ultrastructure of muscle fibers.

Electromyography

Needle EMG is the study of the electrical activity of muscles. EMG may be useful in horses with unusual gait or muscle changes and can be used to discriminate between neurogenic and myogenic causes and more specifically to identify areas or regions of an abnormality.⁷⁵ A recording needle electrode is placed into the muscle, and the electrical activity is amplified, recorded on an oscilloscope, and projected audibly into a loudspeaker. The electrical status of muscle membranes depends on the integrity of the whole motor unit. Thus an EMG evaluates the function of the ventral motor horn cell, its axon, axon terminals, and neuromuscular junctions, as well as the muscle fibers it innervates.

Investigations are usually carried out in two phases. First the practitioner assesses the electrical potentials associated with the physical disruption of the sarcolemma resulting from insertion of the needle. These insertional potentials, if relayed through a loudspeaker, tend to emit a sound similar to short bursts of loud static. They vary among muscles, probably because of differences in the size and number of motor units present.

The second phase involves assessment of electrical potentials when the needle electrode is at rest in the muscle. Normally, electrical silence occurs with cessation of needle movement, unless the needle is located in proximity to a nerve branch or the end plate zone when miniature end plate potentials are recorded continuously; these sound similar to low-intensity static.

In relaxed, diseased muscles, different types of abnormal electrical activity have been recognized. In cases of denervation, insertional activity may persist after needle movement has stopped because of increased excitability of the muscle fiber membranes. Positive, sharp waves are slow monophasic waves, rapid in onset, with a slow decay to the baseline, which occur repeatedly with variable amplitude (100 μV to 20 mV). Their cause is uncertain, they often occur with denervation, and they may represent a nonpropagated depolarization region in the muscle fibers near to the tip of the electrode. When occurring in trains, these positive sharp waves sound similar to a waning brrrr. Fibrillation potentials are electric signals generated by a single muscle fiber. Long, random volleys of mono- or biphasic (occasionally triphasic) potentials of short duration (0.5 to 5.0 ms), with amplitudes of usually less than 200 μV, commonly occur in denervation (depending on the stage). Constant and repetitive fibrillation potentials, which sound similar to rain on a tin roof or the sizzling of frying eggs, can be found especially in the early stages of denervation. Fibrillation potentials can also be seen in myopathic disorders in which segmental muscle necrosis may have caused, for example, the separation of a muscle fiber and its nerve supply.

Myotonic discharges—high-frequency (up to 1000 Hz) repetitive discharges with waxing and waning of potentials is seen and heard with a characteristic, musical, dive-bomber, or more precisely, revving motorcycle–like sound—are found in the myotonias (myotonia congenita, myotonia dystrophica, and hyperkalemic periodic paralysis) if the practitioner moves the exploring EMG electrode or percusses the muscle externally. Bizarre, high-frequency discharges (often called pseudomyotonia) may produce a dive bomber–like sound. No true waxing and waning occurs, although the amplitude and frequency of the potentials may change abruptly to mimic a revving motorcycle–like sound. The discharges are often in couplets or triplets and are likely to terminate abruptly. These, or similar discharges, occur in long-standing denervations, ventral horn cell disease, polymyositis, and certain myopathies. Unlike the myotonic discharges, pseudomyotonic discharges may be abolished by curare and therefore are believed to originate presynaptically.

Fasciculations, visible muscle twitching, are caused by the spontaneous contraction of some or all of the constituent fibers of a motor unit. They can be local or generalized and occur primarily with any cause of muscular weakness (myasthenia), especially in neurogenic disorders such as tetanus and certain debilitating and metabolic disorders. In addition, one can assess the electric activity induced by electric stimulation of nerves or associated with voluntary or induced muscle contraction. Further information on EMG is available.⁷⁶

Thermography

An infrared thermographic scanner converts the radiated thermal energy of the skin to electric signals that can be amplified and displayed on a video screen. By using isotherm colors of known temperature, the examiner can obtain two-dimensional, graphic, and quantitative information regarding the precise temperature of the skin surface. The practitioner can use this technique noninvasively as a means of detecting changes in skin temperature resulting primarily from changes in peripheral blood flow. Inflammation, atrophy, neoplasia, and neurologic lesions, particularly of the autonomic supply to the skin, can alter local blood flow.

Abnormalities have been found in exercise-exacerbated focal thoracolumbar gait abnormalities, as well as disuse atrophy.⁷⁷ The technique has been useful in documenting hindlimb muscle strain as a cause of lameness in horses.

Scintigraphy

Bone-seeking radiopharmaceuticals have been used to detect and localize skeletal muscle involvement, especially in poor performance cases, but are of limited routine value in the field. Accumulation in damaged muscle may be related to the deposition of calcium in damaged fibers, binding by tissue hormones or enzyme receptors, tagging to denatured proteins, or altered capillary permeability. Uptake of labeled phosphates appears to occur only when muscle damage is ongoing and does not occur in areas of repair. Three main types of muscle uptake were identified in one study of horses with skeletal muscle damage: a diffuse, severe, and generalized uptake; bilateral symmetric uptake involving muscle groups that perform synergistic functions; and asymmetric radioisotope uptake in one or more muscle groups on one side of the animal.⁷⁸ The reasons for the various patterns have not been elucidated fully. Scintigraphy can be used as part of a series of tests to identify atypical or difficult-to-pinpoint lamenesses in horses that may have primary muscle lesions among other problems.^79,80

Exercise Tests

Certain physiologic changes can result in a transient alteration in cell membrane permeability. Hypoxia, catecholamines, hypoglycemia, changes in pH, and altered ionic concentrations have been reported as causing such a change in membrane permeability.^45,81,82 Many of these changes are believed to act by decreasing the amount of ATP available for the maintenance of cell integrity, which becomes especially important during exercise.⁸³

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