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Саркомеры — это сократительные единицы мышц, которые расположены в ряд или в виде «коробочного вагона». Вновь синтезированные белки организуются в новые саркомеры и обычно добавляются к концам существующих миофибрилл, что приводит к увеличению длины волокна. Оба типа гипертрофии мышечных волокон требуют синтеза новых белков. Этот процесс включает в себя транскрипцию ДНК в мРНК внутри ядра, транспортировку мРНК из ядра в саркоплазму для трансляции в белок и, наконец, размещение вновь синтезированного белка во всех структурах внутри мышечного волокна. Важно отметить, что этот процесс происходит не только в случае мышечной гипертрофии. На самом деле существует критический и постоянный баланс между синтезом белка и деградацией белка в мышечных клетках.
Белки регулярно собираются, размещаются в клетке и в конечном итоге заменяются вновь синтезированным белком в процессе, известном как белковый обмен. Разборка белков из мышечных волокон опосредована путями деградации белков, которые включают систематический протеолиз белков в пептиды и, в конечном счете, в аминокислоты под действием протеаз. Тремя основными эндогенными протеазными системами в скелетных мышцах являются убиквитиновая протеосома, лизосомальная система и кальций-зависимая протеазная (кальпаиновая) система. Из них кальпаиновая система отвечает за большую часть белкового обмена в живых мышцах, а также является наиболее приемлемым кандидатом на посмертную тендеризацию (протеолиз) мяса. Наиболее хорошо охарактеризованные члены семейства кальпаинов включают μ-калпаин и м-калпаин и их специфический ингибитор кальпастатин.
μ- и m-формы фермента являются цитозольными, повсеместно экспрессируются и требуют микромолярных и миллимолярных концентраций кальция соответственно для активации. Лизосомы представляют собой клеточные органеллы, содержащие протеолитические ферменты, такие как катепсины, которые лучше всего функционируют в среде с низким pH, такой как среда лизосом. Эти ферменты способны расщеплять белки на небольшие пептиды и аминокислоты, но ответственны за относительно небольшой обмен белков в мышцах, отчасти из-за ограниченного количества лизосом, присутствующих в мышцах. Кроме того, роль этой ферментной системы в мышцах еще больше затрудняется тем фактом, что катепсины не могут расщеплять мышечные белки вне лизосом, а лизосомы не могут поглощать интактные миофибриллы. Следовательно, деградация лизосомальной системой в мышцах ограничивается в первую очередь саркоплазматическими белками. Третьей протеазной системой является убиквитиновая протеосома. Протеосома представляет собой большой белковый комплекс, напоминающий полый цилиндр с каталитическим центром, расположенным внутри цилиндра. В отличие от лизосомной системы, протеосомы
сильно сконцентрированы в мышечных клетках, а это означает, что их регулирование имеет решающее значение для предотвращения непреднамеренной деградации. Первая линия регулирования заключается в требовании убиквитинирования. В этом процессе убиквитин ковалентно присоединяется к белкам, что указывает на то, что они должны быть нацелены на деградацию. Затем полиубиквитинированные белки и/или полипептиды транспортируются к протеосоме и предпочтительно расщепляются. Второй контрольной точкой для этой системы является вход в сам цилиндр, который очень мал (1–1,3 нм) в диаметре и ограничивает вход крупных белков или белковых комплексов. Из-за этого ограничения большие белки или комплексы должны быть сначала расщеплены на более мелкие полипептиды с помощью другой протеазной системы, такой как система кальпаина. Исследования показали, что протеосома и кальпаиновая система взаимодействуют для идентификации (нацеливания) и деградации миофибриллярных белков.
Этот процесс включает в себя транскрипцию ДНК в мРНК внутри ядра, транспортировку мРНК из ядра в саркоплазму для трансляции в белок и, наконец, размещение вновь синтезированного белка во всех структурах внутри мышечного волокна. Важно отметить, что этот процесс происходит не только в случае мышечной гипертрофии. На самом деле существует критический и постоянный баланс между синтезом белка и деградацией белка в мышечных клетках. Белки регулярно собираются, размещаются в клетке и в конечном итоге заменяются вновь синтезированным белком в процессе, известном как белковый обмен. Разборка белков из мышечных волокон опосредована путями деградации белков, которые включают систематический протеолиз белков в пептиды и, в конечном счете, в аминокислоты под действием протеаз. Тремя основными эндогенными протеазными системами в скелетных мышцах являются убиквитиновая протеосома, лизосомальная система и кальций-зависимая протеазная (кальпаиновая) система. Из них кальпаиновая система отвечает за большую часть белкового обмена в живых мышцах, а также является наиболее приемлемым кандидатом на посмертную тендеризацию (протеолиз) мяса. Наиболее хорошо охарактеризованные члены семейства кальпаинов включают μ-калпаин и м-калпаин и их специфический ингибитор кальпастатин. μ- и m-формы фермента являются цитозольными, повсеместно экспрессируются и требуют микромолярных и миллимолярных концентраций кальция соответственно для активации. Лизосомы представляют собой клеточные органеллы, содержащие протеолитические ферменты, такие как катепсины, которые лучше всего функционируют в среде с низким pH, такой как среда лизосом. Эти ферменты способны расщеплять белки на небольшие пептиды и аминокислоты, но ответственны за относительно небольшой обмен белков в мышцах, отчасти из-за ограниченного количества лизосом, присутствующих в мышцах. Кроме того, роль этой ферментной системы в мышцах еще больше затрудняется тем фактом, что катепсины не могут расщеплять мышечные белки вне лизосом, а лизосомы не могут поглощать интактные миофибриллы. Следовательно, деградация лизосомальной системой в мышцах ограничивается в первую очередь саркоплазматическими белками. Третьей протеазной системой является убиквитиновая протеосома. Протеосома представляет собой большой белковый комплекс, напоминающий полый цилиндр с каталитическим центром, расположенным внутри цилиндра. В отличие от лизосомной системы, протеосомы сильно сконцентрированы в мышечных клетках, а это означает, что их регулирование имеет решающее значение для предотвращения непреднамеренной деградации. Первая линия регулирования заключается в требовании убиквитинирования. В этом процессе убиквитин ковалентно присоединяется к белкам, что указывает на то, что они должны быть нацелены на деградацию. Затем полиубиквитинированные белки и/или полипептиды транспортируются к протеосоме и предпочтительно расщепляются. Второй контрольной точкой для этой системы является вход в сам цилиндр, который очень мал (1–1,3 нм) в диаметре и ограничивает вход крупных белков или белковых комплексов. Из-за этого ограничения большие белки или комплексы должны быть сначала расщеплены на более мелкие полипептиды с помощью другой протеазной системы, такой как система кальпаина. Исследования показали, что протеосома и кальпаиновая система взаимодействуют для идентификации (нацеливания) и деградации миофибриллярных белков.
Handbook of Meat and Meat Processing Edited by Y. H. Hui, PhD
2.1 Introduction Meat is comprised of numerous tissue types, including nervous, adipose, and connective tissue. Skeletal muscle tissue generally constitutes between 50% and 70% of carcass weight of meat animals, and subsequently, most of the value. Muscle, the living precursor to meat, is a uniquely complex tissue that assumes many forms in order to execute its various functions. Given that muscle is converted into meat postmortem, an understanding of the underlying biology that regulates this tissue antemortem is critical. This chapter will provide a brief introduction to muscle types and introduce the topics of muscle growth and development.
2.2 Types of Muscle The majority of meat derived from carcasses of domestic livestock species is composed of striated skeletal muscle. However, the living animal depends on the functions of three distinct types of muscle to maintain life: smooth, cardiac, and skeletal muscles. These muscles can be distinguished by their primary functions, structures, and nervous control. The following discussion will focus on the properties of skeletal muscle as it is used for food; however, a brief description of these three types of muscle will be given for comparison.
2.2.1 Smooth Muscle Smooth muscle is a critical component of numerous animal tissues providing elasticity to the walls of arteries, linings of the gastrointestinal, reproductive, urinary and respiratory tracts, as well as the lymphatic system. These cells vary widely in their size and shape, but a basic spindle form is common. Smooth muscle cells contain only a single nucleus that is typically located near the center of the cell. As in skeletal muscle, the proteins actin and myosin in smooth muscle are the basis for contraction and movement; however, these proteins are not organized into an ordered, striated appearance as in skeletal muscle. Contractile action of smooth muscle is controlled involuntarily via the autonomic nervous system and therefore conscious thought is not required to generate movement.
2.2.2 Cardiac Muscle Cardiac muscle is located only in the heart where it provides rhythmic contractions throughout an animal’s life. In a similar way to skeletal muscle, cardiac muscle appears striated when viewed under a microscope. However, cardiac muscle contains structural features not observed in skeletal muscle tissue, including branched fibers and intercalated disks, which separate adjacent cardiac muscle cells, and provide the ability to physically communicate contractile force directionally throughout the heart. Similar to smooth muscle, cardiac muscle cells generally contain a centrally located nucleus and are controlled involuntarily by the autonomic nervous system.
2.2.3 Skeletal Muscle Skeletal muscle is the most abundant type of muscle in the animal body. Contractile, structural, and regulatory proteins in this muscle type are highly organized into a distinct striated pattern. Skeletal muscle is so named as it is attached to the skeletal framework of the animal, in various configurations, producing different types of levers and ultimately movement. Skeletal muscle fibers (cells) can range in length from 10 μm to more than a few centimeters. To provide genetic material along the whole length of skeletal muscle fibers, these specialized cells are multinucleated. Nuclei in mature skeletal muscle fibers are located along the periphery of the fiber. Skeletal muscle contraction is directly stimulated by somatic efferent nerves and therefore this muscle type is often referred to as voluntary muscle
2.3 Muscle Growth and Development Although skeletal, smooth, and cardiac muscles take on widely different characteristics and functions in their mature form, early events leading to their development are similar. The development and subsequent growth of muscle is the product of complex cellular changes that accompany myogenesis and the growth of this dynamic tissue pre- and postnatally. To more efficiently produce muscle that results in high-quality meat products, it helps to have an understanding of these events and the processes by which they can be manipulated.
2.3.1 Myogenesis The embryonic cells that ultimately become muscle, with few exceptions, are derived from the mesodermal layer of the developing embryo, a layer that also gives rise to fat and bone tissue. As mesodermal cells become more prominent, they begin to organize into cuboidal clusters known as somites. Further organization of the somites gives rise to the dermomyotome and the sclerotome. Cells of the sclerotome eventually migrate and become the vertebral column while the dermomyotome cells separate to form the dermatome and the myotome, with cells of the dermatome becoming the dermis while cells of the myotome migrate to different regions of the body and give rise to muscle tissue. The expression of one or more genes of a family of transcription factors, referred to as myogenic regulatory factors (MRFs) which are members of the basic helix–loop–helix (bHLH) family of proteins, initiates transcription of muscle-specific genes. This process ultimately results in committing mesodermal cells to the myogenic lineage. As cells express these key genes and become committed to the muscle cell lineage, they are termed myoblasts. Myoblasts are bipolar, spindle-shaped, mononucleated cells that are capable of progressing into the cell cycle and increasing in number through proliferation. However, to complete the developmental process and ultimately produce functioning muscle, cell differentiation must occur for myogenic cells to attain the level of specialization and organization required for contraction. At this point in development, myoblasts are signaled to cease dividing, align with one another, and begin to fuse together to form multinucleated myotubes (immature muscle fibers). Simultaneously musclespecific genes, such as those encoding myosin and actin, are upregulated Four key MRFs responsible for regulating myogenesis are Myf5, MyoD, myogenin, and MRF4. Myf5 and MyoD possess key regulatory functions in the process of myoblast proliferation and differentiation and are more highly expressed in undifferentiated myoblasts. Myf5 is the initial MRF to be expressed in the developing embryo and initiates early myoblast differentiation followed closely by expression of MyoD. Myogenin and MRF4 are considered secondary MRFs as they are responsible for terminal differentiation and maintenance of differentiated myofibrils. Differentiation must occur for the developing cell to become a specialized muscle cell. MRF4 expression occurs later during myogenesis and is the most predominant MRF to be identified in adult skeletal muscle.
2.3.2 Muscle Fiber Formation Muscle fibers express a biphasic developmental pattern in most domestic livestock species, resulting in two distinct populations of fibers, primary and secondary, in the developing fetus. Primary myofibers are generated first, via the fusion of myoblasts, into a structure known as a myotube. Primary myotubes then function as a structure for the organization, alignment, and fusion of other myoblasts into a larger population of secondary myofibers. Primary myofibers form during the initial stage of myogenesis (from approximately months 1–3 postconception in bovine fetuses) while secondary myofibers form during the second wave of myogenesis in the fetal stage (approximately months 3–7 postconception in bovine fetuses Du and others 2010). Histologically, primary myofibers are larger in diameter than secondary myofibers. Both structures initially possess centrally located nuclei; however, in contrast to secondary fibers, primary myofibers express slow myosin ATPase activity. Secondary myofibers maintain communication with their “parent” primary myotubes through membrane-associated proteins that form gap junctions between the structures. However, this association is lost following the innervation and contraction of primary myotubes which elicits the detachment of secondary fibers from the primary. Following separation, a new population of myoblasts can utilize the primary myotube as a template for fusion and development of additional secondary fibers. This well-orchestrated production of secondary myofibers gives rise to approximately 90% of the muscle fibers in most domestic livestock species. It is important to note the role of innervation in this process. Innervation is not required for the formation of primary myotubes. However, because contraction of primary myotubes is required for the development of secondary myofibers, innervation is a controlling factor modulating overall myogenesis. Collectively, these biochemical and morphological events of fetal muscle development occur primarily during the first two-thirds of gestation and for most species are completed by birth. In other words, fiber number is essentially set at birth, with few exceptions. Therefore, events or conditions that enhance or impede embryonic or fetal development can dramatically impact fiber number and ultimate muscle mass of the individual.
2.3.3 Postnatal Muscle Growth Muscle can increase in size by one of three processes: hypertrophy, hyperplasia, or accretion. Hyperplasia refers to an increase in fiber number resulting in an increase in muscle size, while hypertrophy is defined as an increase in fiber size, also resulting in an increase in muscle size. Muscle can also increase in size by the accretion of material, such as fat and connective tissue, between cells; however, this discussion will focus on the mechanisms of hypertrophy and hyperplasia in most domestic livestock species, muscle fiber formation is completed by birth or shortly thereafter and remains essentially stable. Therefore, achieving muscle growth postnatally is dependent on the enlargement of fibers developed prenatally. This hypertrophic growth of muscle fibers is mainly a function of protein synthesis and protein degradation. The difference between the amount of protein synthesized and the amount degraded is termed net protein accretion. During growth, the rate of protein synthesis must exceed the rate of degradation to generate a net accumulation of protein and ultimately an increase in muscle mass. In contrast, when protein degradation exceeds the amount of protein synthesized, muscle atrophy results. The relative rates of synthesis and degradation of muscle proteins influences the longitudinal and radial growth of muscle fibers. Muscle fibers have a diameter of 10–20 µm soon after formation and grow to a normal mature size of 50–80 µm in diameter. To increase in diameter, muscle fibers go through a process known as longitudinal splitting of myofibrils. Myofibrils are the filamentous organelles within muscle fibers, composed of contractile units known as sarcomeres. These organelles split longitudinally to form two new myofibrils which in turn enlarge and become functional via the synthesis of additional contractile, regulatory, and structural proteins. This process of producing new myofibrils is known as myofibrillogenesis. Radial growth of muscle fibers is stimulated by exercise or work and is commonly termed exercise- or workinduced hypertrophy. Conversely, longitudinal growth of muscle fibers is advanced by the stretching of muscles, generally through associated bone lengthening. This type of growth is referred to as stretchinduced hypertrophy and is accomplished through the process of sarcomere addition. Sarcomeres are the contractile units of muscle and are arranged end to end in a series or “box-car fashion.” Newly synthesized proteins are organized into new sarcomeres and generally added to the end of existing myofibrils resulting in an increase in fiber length. Both types of muscle fiber hypertrophy require synthesis of new proteins. This process involves transcribing DNA into mRNA within the nucleus, transporting mRNA out of the nucleus and into the sarcoplasm for translation into protein, and finally arranging the newly synthesized protein into all the structures within the muscle fiber. It is important to note that this process not only occurs in the case of muscle hypertrophy. In fact, a critical and continuous balance exists between protein synthesis and protein degradation in muscle cells. Proteins are regularly assembled, positioned in the cell, and eventually replaced with a newly synthesized protein, in a process known as protein turnover. The disassembly of proteins from muscle fibers is mediated by protein degradation pathways that involve the systematic proteolysis of proteins into peptides and ultimately into amino acids by proteases. The three main endogenous protease systems in skeletal muscle are the ubiquitin proteosome, the lysosomal system, and the calcium-dependent protease (calpain) system. Of these, the calpain system is responsible for a majority of protein turnover in living muscle and is also the most accepted candidate for postmortem tenderization (proteolysis) of meat. The best-characterized members of the calpain family include μ-calpain and m-calpain and their specific inhibitor calpastatin. The μ and m forms of the enzyme are cytosolic, ubiquitously expressed, and require micromolar and millimolar calcium concentrations, respectively, for activation. Lysosomes are cellular organelles containing proteolytic enzymes, such as the cathepsins, that function best in a low-pH environment such as that of a lysosome. These enzymes are capable of degrading proteins into small peptides and amino acids but are responsible for relatively little protein turnover in muscle due, in part, to the limited number of lysosomes present in muscle. In addition, the role of this enzyme system in muscle is further hampered by the fact that cathepsins cannot degrade muscle proteins outside the lysosome and lysosomes cannot engulf intact myofibrils. Therefore, degradation by the lysosomal system in muscle is limited primarily to sarcoplasmic proteins. The third protease system is the ubiquitin proteosome. The proteosome is a large protein complex that resembles a hollow cylinder with the catalytic site residing inside the cylinder. In contrast to the lysosomal system, the proteosome is highly concentrated within muscle cells, which means that its regulation is critical to prevent unintended degradation. The first line of regulation lies with the requirement of ubiquination. In this process, ubiquitin is covalently attached to proteins indicating they are to be targeted for degradation. Polyubiquinated proteins and/or polypeptides are then transported to the proteosome and preferentially degraded. The second control point for this system is the entrance to the cylinder itself, which is very small (1–1.3 nm) in diameter restricting the entrance of large proteins or protein complexes. Because of this restriction, large proteins or complexes must first be degraded into smaller polypeptides by another protease system, such as the calpain system. Research has shown that the proteosome and the calpain system work in cooperation to identify (target) and degrade myofibrillar proteins.
This process involves transcribing DNA into mRNA within the nucleus, transporting mRNA out of the nucleus and into the sarcoplasm for translation into protein, and finally arranging the newly synthesized protein into all the structures within the muscle fiber. It is important to note that this process not only occurs in the case of muscle hypertrophy. In fact, a critical and continuous balance exists between protein synthesis and protein degradation in muscle cells. Proteins are regularly assembled, positioned in the cell, and eventually replaced with a newly synthesized protein, in a process known as protein turnover. The disassembly of proteins from muscle fibers is mediated by protein degradation pathways that involve the systematic proteolysis of proteins into peptides and ultimately into amino acids by proteases. The three main endogenous protease systems in skeletal muscle are the ubiquitin proteosome, the lysosomal system, and the calcium-dependent protease (calpain) system. Of these, the calpain system is responsible for a majority of protein turnover in living muscle and is also the most accepted candidate for postmortem tenderization (proteolysis) of meat. The best-characterized members of the calpain family include μ-calpain and m-calpain and their specific inhibitor calpastatin. The μ and m forms of the enzyme are cytosolic, ubiquitously expressed, and require micromolar and millimolar calcium concentrations, respectively, for activation. Lysosomes are cellular organelles containing proteolytic enzymes, such as the cathepsins, that function best in a low-pH environment such as that of a lysosome. These enzymes are capable of degrading proteins into small peptides and amino acids but are responsible for relatively little protein turnover in muscle due, in part, to the limited number of lysosomes present in muscle. In addition, the role of this enzyme system in muscle is further hampered by the fact that cathepsins cannot degrade muscle proteins outside the lysosome and lysosomes cannot engulf intact myofibrils. Therefore, degradation by the lysosomal system in muscle is limited primarily to sarcoplasmic proteins. The third protease system is the ubiquitin proteosome. The proteosome is a large protein complex that resembles a hollow cylinder with the catalytic site residing inside the cylinder. In contrast to the lysosomal system, the proteosome is highly concentrated within muscle cells, which means that its regulation is critical to prevent unintended degradation. The first line of regulation lies with the requirement of ubiquination. In this process, ubiquitin is covalently attached to proteins indicating they are to be targeted for degradation. Polyubiquinated proteins and/or polypeptides are then transported to the proteosome and preferentially degraded. The second control point for this system is the entrance to the cylinder itself, which is very small (1–1.3 nm) in diameter restricting the entrance of large proteins or protein complexes. Because of this restriction, large proteins or complexes must first be degraded into smaller polypeptides by another protease system, such as the calpain system. Research has shown that the proteosome and the calpain system work in cooperation to identify (target) and degrade myofibrillar proteins.