M. Wick1 and N.G. Marriott2
The Ohio State University Department of Animal Sciences
Researchers continue to study the muscle cell to elucidate its role in meat tenderness. An increased knowledge of sarcomere architecture is essential to understanding the relationship of this contractile unit to muscle growth and development and ultimately meat tenderness. The central event in growth and development of the muscle cell is the precise assembly of the sarcomere, a highly ordered and complex array of numerous proteins. The biological mechanisms controlling the organization of the sarcomere have been extensively studied. This review, excerpted from an invited review article submitted to 1999 Recent Research Advances in Food and Agricultural Chemistry, will discuss the major components in the sarcomere with an emphasis on those components thought to contribute significantly to meat tenderness.
Animal protein excels in palatability, amino-acid balance, and biological value; it is an important nutritional source sought by developed, developing, and undeveloped countries. The harvesting of meat animals with less subcutaneous, intermuscular, and intramuscular fat has necessitated the simultaneous production of more tender musculature, since fat reduction can decrease texture and lubrication during mastication with a resultant perception of decreased tenderness. This production change has necessitated a more thorough knowledge of postmortem changes that occur during the conversion of muscle to meat. Furthermore, an understanding of muscle growth and development is crucial to the efficient production of meat animals for a growing world population that desires a supply of palatable and nutritious protein. Knowledge of sarcomeric architecture is essential to understanding the relationship of this basic contractile unit of the cell to muscle growth, development, and meat tenderness.
Scientists and consumers both recognize that tenderness is one of the most important sensory attributes of meat. The meat industry has responded to consumer desire for tenderness using a variety of techniques including aging, electrical stimulation, mechanical, chemical, and enzymatic tenderization of muscle. Physiologists, muscle-cell biologists, and biochemists continue to investigate the muscle cell to learn more about tenderness indices. In addition, the central event in the growth and development of the muscle cell is the accumulation of the constituent proteins of the sarcomere.
Knowledge of effects of the structural and regulatory proteins of the sarcomere on the physical properties of meat is important to tenderness determinants. These proteins can affect the amount of muscle contraction and sarcomere length, and the structures that they comprise vary in the susceptibility to proteolytic degradation, which ultimately affects tenderness. This review addresses our current understanding of the role of myofibrillar and cytoskeletal proteins in the sarcomere on meat tenderness and quality.
Each skeletal muscle cell is a filamentous, multinucleated structure composed of up to 1,000 fibers termed myofibrils. The sarcomere, the basic contractile unit of muscle (Figure 1), is that portion of the myofibril between two adjacent Z-disks. Photomicrographs suggest that striated muscle sarcomeres are composed of a highly ordered array of two sets of filaments, thin filaments anchored at one end in a structure, the Z-disk, and interdigitating thick filaments (Squire, 1997). The Z-disk is the boundary for the sarcomere and is oriented perpendicular to the thin filaments and the long axis of the myofibril. The thin filaments of the sarcomere, composing the I-band, are isotropic; i.e., they do not refract birefringent light. The thick filaments called A-bands are anisotropic and refract birefringent light (McCormick, 1994). The A-bands are bipolar and possess projections, heads that bind to actin along either end of the filament.
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| Figure 1. Schematic relating the biochemical components to the microscopic structure of muscle tissue. The ultrastructure of the sarcomere showing the myofibrillar composition of muscle segment between two Z-bands. |
The center of the A-band, the "bare-zone," contains a zone free of these projections. The M-line, composed of many "myosin-binding proteins," bisects the bare zone perpendicular to the axis of the sarcomere. The lightly stained zones in the center of the sarcomere, where the thick and thin filaments do not overlap when the sarcomeres are in the relaxed state, are the H-zones. That portion of the H-zone free of thin filaments in the I-band when the sarcomere is fully shortened during contraction is the pseudo-H-zone. Oriented parallel to the Z-disk within the I-band are faint appearing N-lines. A cross section orientation of the sarcomere reveals various patterns of actin, myosin, or actin and myosin filaments. In addition to myofibrillar proteins, the sarcomere contains a third filament system consisting of cytoskeletal proteins that provide lateral and longitudinal support that stabilize the contractile apparatus of the muscle cell, discussed later.
Sarcomeric myosin is an asymmetric molecule, 160 nm in length and 2 to 10 nm wide. Myosin is composed of two 220 kDa heavy chains (MyHCs) and four light chains (LCs) ranging from 17 kDa to 22 kDa (Lowey and Risby, 1971). The entire molecule is approximately 160 nm in length. The heavy chains interact to form two distinct domains; a pair of low-salt-soluble globular heads (S1), 15 nm long and 9 nm wide, and a low-salt-insoluble a-helical coiled-coil rod domain. The rod domain is approximately 150 nm long and 2 nm in diameter and is composed of the C-terminal 1100 amino acids of the MyHC. The myosin rod is a two-stranded coiled-coil motif characterized by two parallel amphipathic a-helical chains that are intertwined into a left-handed superhelix termed an a-helical coiled-coil (McLachlan and Karn, 1982; Crick, 1953)
The globular head or S-1 domain of the MyHC is an actin-activated ATPase responsible for myosin’s molecular motor function. Based on X-ray crystallographic analysis (Rayment et al., 1995), the three-dimensional structure of the S-1 was determined and a significant revision to our understanding of the mechanisms of muscle contraction was proposed (Rayment, 1996).
The C-terminus of the MyHC contains amino-acid sequence elements responsible for the unique solubility and assembly properties of myosin. Furthermore, sequence elements in the C-terminus contribute to the association of a family of myosin-binding proteins (MyBPs). These proteins, along with titin, contribute to the proper assembly of the thick filament. An understanding of these elements will increase our knowledge of the mechanisms controlling muscle growth and development.
Location
Myosin, the most abundant protein in muscle, plays the predominant functional role in many aspects of muscle growth, development, force generation, and tenderness. Recent studies employing molecular genetic techniques have increased our understanding of the contribution of both the N-terminal and the C-terminal sequences in the sarcomeric muscle MyHC to myosin’s unique functions. Elucidation of myosin’s structure/function relationship will contribute not only to technologies that will enable the future production of high-quality processed-meat products but also to an understanding of the biological mechanisms involved in muscle growth, development, and meat tenderness.
Myosin is a protein possessing multiple functions integral to muscle contraction, force generation, muscle development, and production of high-quality processed meats. These functions include Mg++-ATPase activity, molecular motor activity, actin binding, differential salt solubility, thick filament assembly, and intermolecular interactions with constituent proteins in the sarcomere.
Myosin is expressed as a series of developmentally regulated and tissue-specific isoforms or proteins with almost identical structures (Taylor and Bandman, 1989; Gordon and Lowey, 1992; Bandman et al., 1982; Tidyman, 1996; Moore et al., 1992). Different muscles are composed of different proportions of muscle-fiber types. The differential accretion of certain myosin isoforms in specific muscle fiber types is well-documented (Rosser et al., 1997; Rosser et al., 1998; Schiaffino and Salviati, 1997). The functional diversity of myosin isoforms is not understood completely. However, the tenderness and the functionality of meat have been correlated with the proportion of muscle fiber type (Xiong, 1994).
Tenderness
Although the degree of overlap between the thick and thin filaments (Figure 1) contributes significantly to the tenderness of meat, it is the enzymatic degradation of proteins in the Z-band which contributes most significantly to the tenderness of meat. The mechanism of muscle tenderization is not completely understood. Sarcomeres in postmortem muscle that have been aged show disruption of the I-band that is believed to contribute to the tenderization of meat. Calpains, calcium activated proteases, are believed to play a role in the proteolysis of the I-band. In vitro calpains demonstrate specificity for myosin. However, little or nor enzymatic degradation of myosin is known to occur in vivo in postmortem muscle.
Actin
Actin, the predominant protein of the thin filament, along with actin-binding proteins have been identified in organisms across the evolutionary spectrum. The actomyosin complex that forms soon after death is the major contributing feature of rigor mortis. It is the resolution of rigor that contributes to meat tenderness. However, the actomyosin complex is not broken during normal postmortem tenderization or aging. It has not been possible to crystallize the actomyosin complex. However, high-resolution electron microscopy has demonstrated that the actomyosin rigor interface is extensive, involving interaction of a single myosin head with regions on two adjacent actin monomers. A number of hydrophobic residues on the opposing faces of actin and myosin contribute to the main binding site. This site is flanked on three sides by charged myosin surface loops that form predominantly ionic interactions with adjacent regions of actin. Hydrogen bonding is likely to play a significant role in actin-actin and actin-myosin interactions since many of the contacts involve loops (Milligan, 1996).
Z-disks are the boundaries of the sarcomere and aid in maintaining the sarcomere in register in the myofibril. They serve as an anchoring plane of the thin actin filaments; they link titin and actin filaments from opposing sarcomere halves in a lattice connected by a-actinin and contain desmin. Titin, the most abundant and largest muscle cytoskeletal protein, occurs in thin elastic filaments that run parallel to the thick and thin filaments of the sarcomere. Nebulin, another cytoskeletal protein, appears to run parallel to and in close association with the thin filaments. Desmin, a small cytoskeletal protein occurs within and between Z-disks of adjacent sarcomeres to maintain lateral association between sarcomeres. Studies employing protein interaction analysis demonstrate that two types of titin interactions are involved in the assembly of a-actinin into the Z-disk. These scientists indicated that titin interacts by means of a single binding site with the two central spectrin-like repeats of the outermost pair of a-actinin molecules. Spectrin, a cytoskeletal protein composed of a series of repetitive protein motifs, plays a role in maintaining the bi-concave disk morphology of red blood cells. Another function that occurs within the central Z-disk is the interaction of titin with multiple a-actinin molecules by means of their C-terminal domains. This activity permits the assembly of a complex of titin, actin, and a-actinin in vitro with constraint of the path of titin in the Z-disk likely (Young et al., 1998). Actin filaments from adjacent sarcomeres are anchored in the Z-disk of striated muscles. Each filament overlaps with four filaments from the opposite sarcomere, forming a square lattice that is cross-connected in a zigzag pattern by Z-filaments that appear to consist of a-actinin (Luther et al., 1996).
Young et al. (Young et al., 1998) postulated that if titin acted as a regulator for Z-disk assembly, the central Z-disk region of the molecule should contain binding sites for other sarcomeric proteins. This hypothesis is supported by the knowledge that native titin binds to a-actinin (Jeng and S. M. Wang, 1992). In fact, others (Turnacioglu et al., 1996) reported that recombinant fragments of Z-disk titin bind to a-actinin.
The binding of titin to the C-terminal domain of a-actinin appears not to be the only protein interaction that controls the sarcomeric sorting of the molecule (Young et al., 1998). These workers suggest that a second Z-disk specific binding site can be predicted from these observations, which should be important for the proper orientation of the Z-disk. They identified this second binding site of a-actinin as an interaction between the spectrin-like repeats and a single site of titin. Furthermore, they demonstrated that the central Z-repeats of titin can interact equally with the C-terminal domain of a-actinin, similar to the flanking repeats. The assembly of complexes of titin, a-actinin, and actin in vivo are controlled by these interactions. Young and co-workers combined these observations with ultrastructural data on the position of the N-terminus of titin and the C-terminus of nebulin to develop a molecular model of titin, actin, and a-actinin within the Z-disk. This model will be the foundation for studies to more fully understand the architecture of the sarcomere (Young et al., 1998).
Muscle contraction is controlled by the action of a complex of regulatory proteins. In striated muscle, the thin filament consists largely of actin and the actin-binding proteins tropomyosin (Tm) and the troponin (Tn) complex (TnI, TnC, and TnT). The thin filament is responsible for mediating Ca2+ control of muscle contraction and relaxation both antemortem and postmortem. Contraction is initiated by the elevation of the intracellular Ca2+ concentration. Employing EPR and ST-EPR spectroscopy Li et al. proposed that the binding of Ca2+ to TnC induces a series of conformational changes, which ultimately release the inhibition of the actomyosin ATPase activity by TnI (Li et al., 1997). In the absence of Ca2+ binding to troponin C, TM blocks the site of filamentous actin responsible for binding myosin.
Tm is a small polypeptide, (284 amino acids), depending on the particular isoform, and dimerizes to form a head-to-tail coiled-coil structure that lies in the major groove of the actin filament. The placement of the Tm dimer is consistent with one of its important roles, which is to help mediate cooperativity of Ca2+ activation along the length of the myofilament (Solaro and Rarick, 1998). On skeletal muscle thin filaments (Miki et al., 1998), one Tm covers seven actin monomers. Tm is a rigid rod-shaped protein that binds along the length of the actin filament and is intimately associated with TnI. Employing in vitro nanomanipulation, investigators were able to directly measure the stiffness of single actin filaments with and without the presence of Tm and showed that Tm both stabilizes and stiffens the thin filament (Kojima et al., 1994).
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1 For more information, contact at: The Ohio State
University 230A Plumb Hall, 2027 Coffey Road, Columbus, OH 43210, (614) 292-7516,
Fax (614) 292-7116; email:wick.13@osu.edu
2 Department of Food Science and Technology, Virginia Polytechnic
Institute and State University, Blacksburg, Virginia 24061