Modulation of myonuclear number in functionally overloaded and exercised rat plantaris fibers. J. Appl. Physiol. 87(2): [634][635][636][637][638][639][640][641][642] 1999.-The effects of 10 wk of functional overload (FO), with and without daily treadmill endurance training, on the cross-sectional area, myonuclear number, and myonuclear domain size of mechanically isolated single fiber segments of the adult rat plantaris were determined. The fibers were typed on the basis of highresolution gel

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... ectrophoresis for separation of specific myosin heavy chain (MHC) isoforms and grouped as type I ϩ (containing some type I MHC with or without any combination of fast MHCs), type IIa ϩ (containing some type IIa with or without some type IIx and/or IIb but no type I MHC), and type IIx/b (containing only type IIx and/or IIb MHCs). Type I ϩ fibers had a higher myonuclear number than did both fast types of fibers in the control and FO, but not in the FO and treadmill trained, rats. All fiber types in both FO groups had a significantly larger (36-90%) cross-sectional area and a significantly higher (61-109%) myonuclear number than did control. The average myonuclear domain size of each fiber type was similar among the three groups, except for a smaller domain size in the type IIx/b fibers of the FO compared with control. In general, these data indicate that during hypertrophy the number of myonuclei increase proportionally to the increase in fiber volume. The maintenance of myonuclear domain size near control values suggests that regulatory mechanisms exist that ensure a tight coupling between the quantity of genetic machinery and the protein requirements of a fiber. fiber hypertrophy; myosin heavy chains; myonuclear domain size; gel electrophoresis UNLIKE MOST EUKARYOTIC CELLS, skeletal muscle fibers are multinucleated and contain hundreds or even thousands of myonuclei within a common cytoplasm. The reasons for having so many nuclei within a single cell are not well understood but may be related to the enormous size of these cells. The presence of multiple nuclei, therefore, may be a mechanism to avoid diffusion and translocation limitations of nuclear products. Thus each myonucleus may be responsible for the maintenance of a limited volume of cytoplasm, a concept referred to as the "DNA unit" (8) or "myonuclear domain" (15), which is defined as the quantity of cytoplasm regulated by a single myonucleus. The observation that newly transcribed cytoplasmic proteins can be localized to the space around individual myonuclei and neighboring myonuclei in vitro (23, 25) and in vivo (32) gives functional significance to the concept of a myonuclear domain. Myonuclear domain size appears to differ between different fiber types in control animals. Studies in which stereological and morphological techniques were used have shown that, in general, fibers from predominantly slow muscles have a greater myonuclear density than do fibers from predominantly fast muscles in rats (4, 6), rabbits (6), and chickens (19) . More recently, confocal microscopy was used to demonstrate that slow fibers from the plantaris muscle of control rats (37) and cats (2) have 25-50% more myonuclei per millimeter and possess a smaller average myonuclear domain size than do fast fibers. The reason for the difference in myonuclear domain size between slow and fast fibers is not currently known but may be related to the greater quantity of enzyme synthesis necessary in more highly oxidative fibers (37) and/or the greater protein turnover level of slow vs. fast muscles (5). In addition, it is also unclear whether these relationships are maintained within distinct adult fiber types during muscle adaptation. During muscle fiber hypertrophy, one cellular strategy for increasing protein synthesis would be to increase RNA synthesis from existing myonuclei. In this scenario, myonuclear domain size would increase as each myonucleus encoded more proteins for a larger volume of cytoplasm. An alternative strategy would be to maintain the level of RNA synthesis per myonuclei at a normal level but to increase the amount of DNA available to the cell by adding more myonuclei. In this scenario, myonuclear domain size would remain unchanged or even decrease. Evidence to date has strongly suggested that fiber hypertrophy is accompanied by commensurate changes in myonuclear number such that myonuclear domain size remains unchanged (2, 20). However, whether this is true for different types of fibers within a muscle that has a mixture of fiber types is not currently known. It is generally assumed that satellite cells provide a source for new myonuclei in adult skeletal muscle (9, 22, 33) , and it has been suggested that their fusion with the fiber may be crucial to the hypertrophic response (26). In particular, these latter studies have demonstrated that, if cell proliferative activity is ablated by using irradiation, increases in myonuclear number and fiber hypertrophy do not occur (26, 27) . These data indicate that the increase in fiber volume during fiber hypertrophy appears to be dependent on the modulation of myonuclear number via satellite cell activation. What is less clear is whether there is any limit to the response of muscle stem cells in producing new myonuclei. In a previous study, myonuclear number increased The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 8750-7587/99 $5.00