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This comparative study of myonuclear domain (MND) size in mammalian species representing a 100 000-fold difference in body mass, ranging from 25 g to 2500 kg, was undertaken to improve our understanding of myonuclear organization in skeletal muscle fibres. Myonuclear domain size was calculated from three-dimensional reconstructions in a total of 235 single muscle fibre segments at a fixed sarcomere length. Irrespective of species, the largest MND size was observed in muscle fibres expressing fast myosin heavy chain (MyHC) isoforms, but in the two smallest mammalian species studied (mouse and rat), MND size was not larger in the fast-twitch fibres expressing the IIA MyHC isofom than in the slow-twitch type I fibres. In the larger mammals, the type I fibres always had the smallest average MND size, but contrary to mouse and rat muscles, type IIA fibres had lower mitochondrial enzyme activities than type I fibres. Myonuclear domain size was highly dependent on body mass in the two muscle fibre types expressed in all species, i.e. types I and IIA. Myonuclear domain size increased in muscle fibres expressing both the β/slow (type I; r= 0.84, P < 0.001) and the fast IIA MyHC isoform (r= 0.90; P < 0.001). Thus, MND size scales with body size and is highly dependent on muscle fibre type, independent of species. However, myosin isoform expression is not the sole protein determining MND size, and other protein systems, such as mitochondrial proteins, may be equally or more important determinants of MND size.
Skeletal muscle is a highly ordered structure composed of muscle cells that can be several centimetres long. In order to support the large cytoplasm, skeletal muscle fibres are one of the few truly multinucleated types of cells. Each cell can encompass several hundred myonuclei, and each myonucleus regulates the gene products in a finite volume of a muscle fibre, i.e. the myonuclear domain (MND) or DNA unit. The term ‘myonuclear domain’ has been defined as the theoretical volume of cytoplasm associated with a single myonucleus (Cheek, 1985; Hall & Ralston, 1989; Allen et al. 1995, 1999). This concept originates from the finding that mRNA produced by a single nucleus is confined to the area immediately surrounding that particular nucleus (Ralston & Hall, 1992), although the proteins encoded by a single nucleus may be found along the length of a myofibre (Ralston & Hall, 1992). It is important to recognize that the regulation of expression and distribution of individual proteins within the muscle fibre is dependent on a number of variables related to the nature of each protein. For instance, cytoplasmic or membrane proteins are free to diffuse and occupy the whole length of the fibre. In contrast, if proteins are associated with a target, their distribution will be restricted. For example, acetylcholine receptor, a protein that is localized in the postsynaptic membrane of the neuromuscular junction, is concentrated near myonuclei at the motor endplate rather than distributed throughout the muscle fibre (Merlie & Sanes, 1985).
It is generally assumed that postnatal muscle growth occurs exclusively through an increase in myofibre size without an increase in myofibre number, and satellite cells provide a source for new myonuclei at a rate sufficient to maintain a constant myonuclear domain size during normal skeletal muscle growth (Moss, 1968; Schultz, 1989). When a skeletal muscle is subjected to increased mechanical stress, either by regular exercise or by functional overload, it results in increases in muscle mass, fibre size and the number of myonuclei (Roy et al. 1985, 1991). The increase in fibre size is thought to occur via multiple mechanisms, including changes in gene transcription and the rate of protein synthesis. The number of myonuclei has been reported to increase during hypertrophy of adult muscle fibres (Allen et al. 1995), i.e. a proportional increase in myonuclear number and fibre size that results in a constant MND size of skeletal muscle fibres during muscle fibre hypertrophy (Rosenblatt et al. 1994; Roy et al. 1999; Sinha-Hikim et al. 2003). This causal link between satellite cell proliferation, increased myonuclear number, increased fibre sizes and a constant MND size during muscle hypertrophy has been well recognized, but there are also reports of skeletal muscle hypertrophy without addition of satellite cells (Schultz, 1989). Furthermore, there are also divergent reports regarding changes in MND size during muscle atrophy, i.e. a decreased, unaltered or increased total number of myonuclei have been reported in combination with decreased muscle fibre size. By using a number of muscle atrophy models, Allen et al. (1995, 1996, 1997) demonstrated that both fibre size and myonuclear number decreased, and that fibre size was consistently decreased to a greater extent than myonuclear number, resulting in a considerable decrease in MND size. Kasper & Xun (1996b), in contrast, reported an increased number of myonuclei and decreased MND size during the muscle wasting associated with hindlimb suspension. Finally, time-resolved in vivo analyses of the number myonuclei have shown that disuse atrophy is not accompanied by a loss of the number of myonuclei during the first 2 months of denervation atrophy (Bruusgaard & Gundersen, 2008).
The cytoplasm-to-myonucleus ratio appears to be related to muscle fibre myosin heavy chain (MyHC) isoform expression and to the amount and/or rate of protein synthesis and degradation, with the MND size of fibres expressing the β/slow (type I) MyHC (MyHCI) isoform being smaller than that of fibres expressing fast MyHCs (types IIA, IIX and IIB) isoforms (Tseng et al. 1994; Kasper & Xun, 1996a; Allen et al. 1999; Roy et al. 1999). This is also true when MND size is compared between muscles with slow and fast profiles from different species, i.e. a smaller MND size has been reported in muscle fibres from slow than from fast muscles in the rat (Burleigh, 1977; Atherton & James, 1980), rabbit (Burleigh, 1977) and chicken (Matthew & Moore, 1987). Although it remains unclear why MND size should be smaller in slow oxidative than in fast glycolytic fibres, it has been hypothesized that the higher concentration of myonuclei per unit cytoplasmic volume in slow fibres is related to their higher rate of protein turnover (Edgerton & Roy, 1991; Tseng et al. 1994).
Our understanding of muscle structure and function is, in general, based on observations in small rodents and amphibians. The spatial organization of nuclei in human muscle cells in health and disease is poorly understood, partly because there are insufficient methods to identify and characterize the spatial arrangement of myonuclei in three dimensions. The situation is further complicated by the fact that MND size varies depending on the species and MyHC isoform expression. In spite of the significant similarities in regulation of muscle contraction at the cellular and motor protein levels between different species, there are also significant differences. More than half a century ago, A. V. Hill (Hill, 1950) postulated that contractile properties of skeletal muscle differ between species owing to differences in body mass or limb length, since animals of similar body shape and gait characteristic move at similar velocities independent of body size. The mechanisms underlying species differences in regulation of muscle contraction have been studied at the muscle fibre and motor protein levels (Rome et al. 1990; Rome, 1992; Höök et al. 2001; Pellegrino et al. 2003; Andruchov et al. 2004; Bicer & Reiser, 2004, 2007; Marx et al. 2006), but our understanding of MND size in different muscle fibre types in mammalian species representing a large body size range is incomplete. There is, accordingly, a compelling need for comparative studies on differences in myonuclear organization between species, including humans, that represent a large body mass range and to transfer this knowledge into clinical research focusing on the mechanisms underlying human muscle wasting in different disease states.
This study aimed to test the hypothesis that MND size is dependent on both MyHC isoform expression and body mass, and muscle tissue has been analysed from mammalian species representing a 100 000-fold difference in body size, i.e. muscle fibres from mouse, rat, human, pig, horse and rhinoceros muscles.