Which one of the following is called a sarcomere in myofibril?

1 Introduction

Sarcomere, which is the basic unit of striated muscles, is a complex multicomponent biological system controllably transforming the chemical energy released upon ATP hydrolysis into mechanical work. Since the change in sarcomere length in a contracting muscle requires the presence of well-coordinated moving parts, many muscle proteins evolved to quickly adapt to the variable molecular environment and geometry.1 Consequently, protein regions that do not possess a rigid three-dimensional structure are expected to play important roles in the sarcomere functions.

Protein components and architecture of the sarcomere have been extensively reviewed, and the interested reader is directed to a review by Henderson et al.2 for more comprehensive information on the subject. Here, we will mainly focus on functionalities of intrinsically disordered regions (IDRs) in two key muscle protein groups within the context of the sarcomere structure and operation (Fig. 1). One protein group, comprising skeletal and cardiac isoforms of myosin-binding protein C (MyBP-C), is recognized as having a regulatory role in sarcomere contraction.2–4 Another protein group consists of several isoforms of tropomodulin/leiomodin homology family, and it is known to regulate thin filament formation.2,5–7 Both groups represent two different yet strongly related aspects of the sarcomere function, namely its structure and how this structure enables normal (or abnormal, if in disease) muscle performance in a constantly changing environment.

The Sarcomere: The Basic Contractile Unit of Muscle

A sarcomere is the basic contractile unit of muscle fiber. Each sarcomere is composed of two main protein filaments—actin and myosin—which are the active structures responsible for muscular contraction. The most popular model that describes muscular contraction is called the sliding filament theory. In this theory, active force is generated as actin filaments slide past the myosin filaments, resulting in contraction of an individual sarcomere.

Fig. 3.5 illustrates a sarcomere and emphasizes the physical orientation of the actin and myosin filaments. The thick myosin filament contains numerous heads, which when attached to the thinner actin filaments create actin-myosin cross bridges. In essence, a myosin head is similar to a cocked spring, which on binding with an actin filament flexes and produces a power stroke. The power stroke slides the actin filament past the myosin, resulting in force generation and shortening of an individual sarcomere (Fig. 3.6). Because sarcomeres are joined end to end throughout an entire muscle fiber, their simultaneous contraction shortens the entire muscle.

Each myosin filament has numerous heads, and each actin filament has numerous binding sites. This is important because in order for a sarcomere to maximally contract, numerous power strokes must occur. In fact, the force of a muscular contraction is determined largely by the number of actin-myosin cross bridges that are formed. This concept is addressed later in the section on the importance of muscular length.

Importance of the Sarcomere

To understand the SL–tension relationship, it is important to understand the sarcomere. The sarcomere is the fundamental unit of myocyte contraction. Sarcomeres are recognizable as the familiar banding pattern observed when striated muscle is seen through the light microscope. Figure 3(a) shows a portion of a ventricular myocyte from a bluefin tuna where the regular banding pattern of the sarcomeres is clearly visible. A schematic of a mammalian sarcomere and its composite proteins is provided in Figure 3(b). The morphology of the rainbow trout sarcomere is similar to that of the mammalian sarcomere and thin-filament length is approximately 0.95 μm in both rat and rainbow trout ventricular myocytes. A sarcomere is defined as the distance between the Z-lines. The Z-lines are pulled closer together during contraction and move further apart during relaxation. The Z-lines are closer during contraction because actin and myosin interaction generates cross-bridges, which slide the myofilaments over each other. During relaxation, myosin and actin detach and the Z-lines slide back apart. The role of myofilament overlap in sarcomere shortening is expanded upon in the following section (see also DESIGN AND PHYSIOLOGY OF THE HEART | Cardiac Excitation–Contraction Coupling: Calcium and the Contractile Element).

miRNA Regulation of Cardiomyocyte Sarcomere Organization

Sarcomeres are the basic contractile units of cardiac muscle. They are composed of thick and thin filaments essential for generation and propagation of mechanical force. Myosin, the major component of the thick filament, is comprised of MHC subunits and myosin light-chain (MLC) subunits. α-MHC (Myh6) and β-MHC (Myh7) are both expressed in the heart during development and in the adult. In rodents, expression of β-MHC is downregulated after birth so that in the adult, α-MHC is the dominant MHC isoform in the heart (Lyons et al., 1990; England and Loughna, 2013). MHC isoform switches in response to cardiac stress or hypothyroidism. Pathologic hypertrophy is associated with upregulation of β-MHC and downregulation of α-MHC (Krenz and Robbins, 2004; Gupta, 2007).

Expression of α- and β-MHC isoforms is controlled by miRNA-208a, miRNA-208b, and miRNA-499 (van Rooij et al., 2007, 2009; Callis et al., 2009). miRNA-208a and miRNA-208b are encoded in an intron of the α-MHC and β-MHC gene, respectively. Mice null for miRNA-208a are viable but show abnormalities in sarcomere structure and declined cardiac function at 6 months of age (van Rooij et al., 2007). However, miRNA-208a null mice are resistant to cardiac hypertrophy in response to stress induced by transverse aortic banding or calcineurin (van Rooij et al., 2007; Callis et al., 2009). This is concomitant with decreased expression of slow skeletal muscle contractile protein, β-MHC, in the miRNA-208a null heart. The function of miRNA-208a is mediated, in part, by repression of thyroid hormone receptor associated protein 1 (Thrap1), which negatively regulates the expression of β-MHC.

miRNA-208a controls not only the expression of β-MHC in the heart but also that of the closely related slow myosin isoform, Myh7b (van Rooij et al., 2009). Both β-MHC and Myh7b genes encode intronic miRNAs, miRNA-208b, and miRNA-499, respectively (Berezikov et al., 2006; Landgraf et al., 2007). Mice lacking the miRNA-208b or miRNA-499 gene have no obvious developmental defects (van Rooij et al., 2009). However, miRNA-208b/-499 double null mutant mice display reduced expression of slow myofiber β-MHC and increased expression of fast type myosin isoforms. By contrast, overexpression of miRNA-499 leads to increased expression of β-MHC and drives muscle toward a slow myofiber phenotype. Forced cardiac miRNA-499 expression promotes hypertrophy in mice (Shieh et al., 2011; Matkovich et al., 2012). Together, these miRNAs are important in the specification of the identity of muscle fibers by stimulating slow myofiber gene programs at the expense of those that control fast myofiber gene expression (Hodgkinson et al., 2015).

It has been demonstrated that miRNA-1 and miRNA-133 can act as specific activators or suppressors of sarcomere formation and muscle gene expression. Deletion of both miRNA-1-2 and miRNA-1-1 in mice (miRNA-1 null) leads to sarcomere disruption in cardiomyocytes and impaired cardiac function. All the miRNA-1 null mice died before weaning age (Heidersbach et al., 2013; Wei et al., 2014). miRNA-1 functions to negatively regulate myocardin, the major regulator of smooth muscle gene expression, and telokin, the smooth muscle-specific inhibitor of MLC-2 phosphorylation (Heidersbach et al., 2013; Wystub et al., 2013). The upregulation of myocardin and telokin in miRNA-1 null hearts may, in part, contribute to the defect in sarcomere organization. Furthermore, studies from Wei et al. indicated that miRNA-1 directly represses nuclear receptor estrogen-related receptor β (Errβ). The increased level of Errβ in miRNA-1 null heart activates the expression of fetal sarcomere-associated genes (Wei et al., 2014).

miRNA-133a represses smooth muscle gene expression in the heart by directly targeting myocardin and SRF for repression (Liu et al., 2008; Wystub et al., 2013). Deletion of both miRNA-133a-1 and mIRNA-133a-2 (miRNA-133a null) causes late embryonic and neonatal lethality due to ventricular septal defect (VSD) and chamber dilation (Liu et al., 2008). miRNA-133a null mice display sarcomere disorganization and ectopic activation of the smooth muscle gene program (Liu et al., 2008). In addition, mice lacking both miRNA-1 and miRNA-133a displayed severe cardiac dysfunction and died before embryonic day 11.5 (E11.5). Mice with a null mutation in miRNA-1/133a showed increased expression of myocardin and smooth muscle genes in the heart. These studies indicate that miRNA-1 and miRNA-133a clusters are important in cardiomyocyte differentiation and sarcomere formation during embryonic and postnatal life. They act cooperatively to govern the gene transition program from an immature state characterized by expression of smooth muscle genes to a mature phenotype (Wystub et al., 2013).

Myocardial energetics

Sarcomere dysfunction has been considered the defect in the pathogenesis of HCM. However, the discovery of PRKAG2 mutation, a nonsarcomeric gene, raised the possibility that defective myocardial energetics may be a common feature of HCM mutations. Alterations of myocardial metabolism had been observed in human HCM14 and in α-MHC403/+ mice.15,16 Although these energetic changes might result from factors such as myocardial ischemia due to increased oxygen demands of the hypertrophied myocardium, it is also possible that alterations of myocardial metabolism may occur as primary effects of mutant proteins. Moreover, mutations that cause deficiency in mitochondrial ATP production (e.g., mtDNA mutations) can also result in HCM (see later in this chapter).

Sarcomeres

A sarcomere is the functional unit (contractile unit) of a muscle fiber. As illustrated in Figure 2-5, each sarcomere contains two types of myofilaments: thick filaments, composed primarily of the contractile protein myosin, and thin filaments, composed primarily of the contractile protein actin. Thin filaments also contain the regulatory proteins, troponin and tropomyosin. When myofilaments are viewed under an electron microscope, their arrangement gives the appearance of alternating bands of light and dark striations. The light bands are called I bands and contain only thin filaments. The dark bands are called A bands and contain thick and thin filaments, with the thick filaments running the entire length of the A band. Thus the length of the thick filament determines the length of the A band.

The names for the various regions of the sarcomere are not arbitrary; they are derived from the first letter of the German word that describes their appearance. The names for the bands describe the refraction of light through the respective bands. The I band is abbreviated from the word isotropic, which means that this area appears lighter because more light can pass through it. The A band is so named because of its anisotropic properties, meaning that it appears darker because it does not allow as much light to pass through. These properties are directly related to the type of filament present.

Each A band is interrupted in the midsection by an H zone (from the German Hellerscheibe, for “clear disc”), where there is no overlap of thick and thin filaments. Running through the center of the H zone is a dense line called the M line (from the German Mittelsclzeibe, for “middle disc”). The I bands are also interrupted at the midline by a darker area called the Z disc (from the German Zwischenscheibe, for “between disc”). A sarcomere extends from one Z disc to the successive Z disc. The Z disc serves to anchor the thin filaments to adjacent sarcomeres.

Myofilaments occupy three-dimensional space. The arrangement of the myofilaments at different points in the sarcomere is shown in Figure 2-5, D and F. Notice that in regions where the thick and thin filaments overlap, each thick filament is surrounded by six thin filaments and each thin filament is surrounded by three thick filaments.

A sarcomere consists of more than just contractile and regulatory proteins. Proteins of the cytoskeleton provide much of the internal structure of the muscle cell. Figure 2-6 diagrams the cytoskeleton of the sarcomere and its relationship to the contractile proteins.6 The M line and the Z disc hold the thick and the thin filaments in place, respectively. The elastic filament helps keep the thick filament in the middle between the two Z discs during contraction.

Muscle Contraction Involves the Sliding of the Thick and Thin Filaments Relative to Each Other in the Sarcomere

Measurements of sarcomere and A and I band lengths from electron micrographs of contracted and resting muscle firmly established the mechanism of muscle contraction: The sliding of actin thin and myosin thick filaments passed each other within the sarcomere unit. These measurements demonstrated that the lengths of the individual filaments do not change as a muscle contracts; yet, the distance between two adjacent Z disks becomes shortened in contracted muscle relative to relaxed muscle. When the length of a sarcomere decreases in contracted muscle, the I band region shortens, whereas the length of the A band remains unchanged (Fig. 3-10).

Because the lengths of the thick and thin filaments do not change, the change in length of the I band could occur only if the thin filaments were to slide past the thick filaments. Therefore, the reversed polarity of the thick and thin filaments relative to the center line of the sarcomere (defined by the M line) would cause a shortening of the sarcomere during contraction by the sliding of thin actin filaments, which are attached to the Z disk, past the thick myosin filaments toward the center of the sarcomere. This model of muscle contraction, called the sliding filament model, was first proposed in 1954 and led to the dissection of molecular mechanisms of contraction.

Alterations in the Myofilament (see also Chapter 3)

Sarcomere shortening is the result of cyclic cross-bridge formation between the myosin “head” formed by the myosin heavy chain (MHC) and active sites on actin. The process is regulated by the thin filament associated proteins, troponin (Tn) C, I, and T, and tropomyosin. Ventricular myocardium is composed of two MHC isoforms, α and β. Normal rodent myocardium is composed predominantly of α-MHC while normal human myocardium is predominantly β-MHC.99 Major increases in the proportion of the β-MHC isoform are observed in rodent models of HF and STZ DM.100,101 Compared with α-MHC, β-MHC exhibits slower kinetics as it interacts with actin. This myosin isoform shift likely contributes to the depressed contractile parameters observed in STZ DM. Since nonfailing human myocardium is composed of predominantly β-MHC, any myosin isoform shift toward the β isoform in patients with DM would be small and thus not likely to be functionally important.99 In STZ rats, an increase in TnI phosphorylation has been reported, which may affect contractile protein function.102 In humans, maximal myofilament contractile force and calcium sensitivity is decreased in detergent-treated (skinned) myocardial strips obtained from patients with DM.103 These preparations are used to assess myofilament function independent of changes in calcium cycling. The changes in myofilament function in this human study were associated with an increase in TnI and TnT phosphorylation. TnI and TnT phosphorylation have complex effects on myofilament contractile function67 and could also contribute to impaired ventricular contraction and relaxation. Finally, depressed optimal frequency of work production and power generation in skinned strips from female but not male coronary artery bypass patients with DM has been reported.104 This could possibly help explain poorer outcomes in women with HF and DM.

As with calcium cycling proteins, posttranslational modification of myofilament proteins by ROS and AGEs may also contribute to myofilament dysfunction. In this regard, in addition to detecting AGEs in association with collagen fibrils, we have also identified AGEs in myofilament sections in biopsy specimens from patients with DM (see Figure 26-5, right panel).

47.3.5.2.2 β-Myosin Heavy Chain (MYH7)

This sarcomere protein was first implicated in DCM via genetic linkage studies of a large family with DCM that mapped to chromosome 14q (156). A β-myosin heavy chain missense mutation was identified in that family and subsequently in other DCM families that altered residues that have not been implicated in HCM. Biophysical analyses of DCM mutations in β-myosin heavy chain (e.g. Ser532Pro) indicate that these can diminish force generation by disruption of actomyosin binding. In contrast, another DCM mutations (e.g. Phe764Leu) located in the head-neck hinge region of myosin alters the mechanics of contractile efficiency or force transmission (157).

Sarcomeres and Costameres

The sarcomere is the basic structural and functional unit of the fibril. It is bordered by a Z-band on each end with adjacent I-bands, and there is a central M-line with adjacent H-bands and partially overlapping A-bands. The Z-band (or Z-disk) is a dense fibrous structure made of actin, α-actinin, and other proteins. Thin filaments (or actin filament) are anchored at one end at the Z-band. Titin is anchored to both the Z-band and the M-line. Thick filaments are anchored in the middle of the sarcomere at the M-line. The I-band is the region on either side of a Z-disc that contains only thin filaments and titin. This partial overlap in filaments makes the A-band darker at its ends, leaving a light area in the middle (H-band) where there is no overlap with the light bands. A key clue to the mechanism of contraction was the finding that during contraction the H and I bands shorten, while the A bands do not.

The sarcolemma has more-or-less regularly spaced electron-dense patches or rings of large membrane-spanning multiunit proteins. These are analogous to desmosomes, and the proteins therein are structural proteins linking the meshwork of cytoskeletal filaments that tie the myofibrils together to the extracellular matrix. The complex of cytoskeletal actin and IFs and the membrane proteins is called a costamere.

Desmin, an intermediate filament protein, forms a network from one Z-disk to the next across the myofibril. These networks are linked by actin, via the protein dystrophin, to the sarcolemma. This costamere structure helps hold the sarcomeres in register and plays an important role in transmitting force produced in the myofibrils to adjacent myofibrils and, via the transmembrane components of the costamere, to the extracellular matrix. This mechanism is the major means of force transmission from myofibril to tendon. Duchenne and Becker’s muscular dystrophy are due to mutations in, or deletion of, the dystrophin gene.