Yoko Higo

Essential role of satellite cells in the growth of rat soleus muscle fibers

Effects of gravitational loading or unloading on the growth-associated increase in the cross-sectional area and length of fibers, as well as the total fiber number, in soleus muscle were studied in rats. Furthermore, the roles of satellite cells and myonuclei in growth of these properties were also investigated. The hindlimb unloading by tail suspension was performed in newborn rats from postnatal day 4 to month 3 with or without 3-mo reloading. The morphological properties were measured in whole muscle and/or single fibers sampled from tendon to tendon. Growth-associated increases of soleus weight and fiber cross-sectional area in the unloaded group were approximately 68% and 69% less than the age-matched controls. However, the increases of number and length of fibers were not influenced by unloading. Growth-related increases of the number of quiescent satellite cells and myonuclei were inhibited by unloading. And the growth-related decrease of mitotically active satellite cells, seen even in controls (20%, P > 0.05), was also stimulated (80%). The increase of myonuclei during 3-mo unloading was only 40 times vs. 92 times in controls. Inhibited increase of myonuclear number was not related to apoptosis. The size of myonuclear domain in the unloaded group was less and that of single nuclei, which was decreased by growth, was larger than controls. However, all of these parameters, inhibited by unloading, were increased toward the control levels generally by reloading. It is suggested that the satellite cell-related stimulation in response to gravitational loading plays an essential role in the cross-sectional growth of soleus muscle fibers.

The Role of Neural and Mechanical Influences in Maintaining Normal Fast and Slow Muscle Properties

The relative importance of neural and mechanical influences in maintaining normal slow and fast muscle properties remains unclear. To address this issue, we studied the effects of 10 days of hindlimb unloading (HU) with or without tenotomy and/or denervation on the cross-sectional area (CSA), myosin heavy chain (MHC) expression (immunohistochemistry) and composition (gel electrophoresis), and myonuclear number in soleus and plantaris fibers in adult male Wistar rats. In general, the adaptations in fiber type and size were similar using either single fiber gel or immunohistochemical analyses. HU resulted in atrophy of type I and I+IIa/x MHC fibers in the soleus and in type I, I+IIa/x, IIa/x, IIa/x+IIb, and IIb MHC fibers in the plantaris. Addition of tenotomy and/or denervation in HU rats had minimal effects on fiber CSA in the soleus, but fiber CSA in the plantaris further decreased, particularly in fibers expressing only fast MHCs. HU resulted in a de novo appearance of type I+IIa/x+IIb and IIa/x+IIb MHC fibers in the soleus and of type I+IIa/x+IIb MHC fibers in the plantaris.Tenotomy and/or denervation in HU rats had no further effect on the fiber type composition of either muscle. Mean myonuclear number/mm of type I fibers was decreased in the soleus of HU rats, and increased in type I and I+IIa/x fibers in HU plus tenotomy (HU+Ten) rats. In the plantaris, mean myonuclear number/mm of type IIa/x, IIa/x+IIb, and IIb fibers was lower after HU with or without tenotomy and/or denervation. Mean cytoplasmic volume/myonucleus ratio of type I and I+IIa/x fibers in the soleus of the HU group tended to be smaller than in controls. The largest decrease was noted in the HU+Ten group. In the plantaris, this ratio was unaffected by HU alone, but was decreased by addition of tenotomy and/or denervation when all fiber types were combined. These data indicate that the major cause of fiber atrophy and adaptations in myonuclear domain size in the slow soleus of HU rats is the chronic reduction in force generation, whereas the elimination of neuromuscular contact via denervation results in additional fiber atrophy and adaptations in myonuclear domain size in the fast plantaris.

Myonucleus-Related Properties in Soleus Muscle Fibers of mdx Mice

Distribution and total number of myonuclei in single soleus muscle fibers, sampled from tendon to tendon, were analyzed in mdx and wild-type (WT) mice. Apoptotic myonuclei and the microscopic structure around the myonuclei were also analyzed. Three types of muscle fibers of mdx mice with myonuclear distribution at either central, peripheral, or both central and peripheral regions were observed in the longitudinal analyses. All of the myonuclei were located at the peripheral region in WT mice. The total number of myonuclei counted in the whole length of fibers with peripheral myonuclei only was 17% less in mdx than in WT mice (p < 0.05). But the total myonuclear numbers in mdx mouse fibers with different distribution (peripheral vs. central) of myonuclei were identical, and the peripheral nucleus was noted where the central nucleus was missing. Myonuclei located between the center and peripheral regions were also seen in the cross-sectional analyses of muscle fibers. The cross-sectional area and length of fibers, sarcomere number, myonuclear size, myosin heavy chain expression, satellite cell number and neuromuscular junction were identical between each type of fiber. Apoptosis was not detected in any myonuclei located either in central or peripheral regions of muscle fibers. Thus, it was suggested that apoptosis-related loss of central myonuclei and regeneration-related new accretion at the peripheral region is not the cause of different distribution of myonuclei seen in muscle fibers in mdx mice. However, it was speculated that cross-sectional migration of myonuclei from central to peripheral regions may be induced in response to regeneration, because the total myonuclear numbers in fibers with different distribution of myonuclei were identical, and the peripheral nucleus was noted where the central nucleus was missing. Further, myonuclei located between the center and peripheral regions were also seen. However, the question remains as to how or why nuclei might migrate to the periphery in a regenerating muscle fiber, since there was no microscopic evidence of any structural changes around the myonuclei that may be responsible for the movement of the nucleus.

Effects of Creatine and Its Analog, β-Guanidinopropionic Acid, on the Differentiation of and Nucleoli in Myoblasts

The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.