Mechanisms leading to restoration of muscle size with exercise and transplantation after spinal cord injury.

We have shown that cycling exercise combined with fetal spinal cord transplantation restored muscle mass reduced as a result of complete transection of the spinal cord. In this study, mechanisms whereby this combined intervention increased the size of atrophied soleus and plantaris muscles were investigated. Rats were divided into five groups (n = 4, per group): control, nontransected; spinal cord transected at T10 for 8 wk (Tx); spinal cord transected for 8 wk and exercised for the last 4 wk (TxEx); spinal cord transected for 8 wk with transplantation of fetal spinal cord tissue into the lesion site 4 wk prior to death (TxTp); and spinal cord transected for 8 wk, exercised for the last 4 wk combined with transplantation 4 wk prior to death (TxExTp). Tx soleus and plantaris muscles were decreased in size compared with control. Exercise and transplantation alone did not restore muscle size in soleus, but exercise alone minimized atrophy in plantaris. However, the combination of exercise and transplantation resulted in a significant increase in muscle size in soleus and plantaris compared with transection alone. Furthermore, myofiber nuclear number of soleus was decreased by 40% in Tx and was not affected in TxEx or TxTp but was restored in TxExTp. A strong correlation (r = 0.85) between myofiber cross-sectional area and myofiber nuclear number was observed in soleus, but not in plantaris muscle, in which myonuclear number did not change with any of the experimental manipulations. 5'-Bromo-2'-deoxyuridine-positive nuclei inside the myofiber membrane were observed in TxExTp soleus muscles, indicating that satellite cells had divided and subsequently fused into myofibers, contributing to the increase in myonuclear number. The increase in satellite cell activity did not appear to be controlled by the insulin-like growth factors (IGF), as IGF-I and IGF-II mRNA abundance was decreased in Tx soleus and plantaris, and was not restored with the interventions. These results indicate that, following a relatively long postinjury interval, exercise and transplantation combined restore muscle size. Satellite cell fusion and restoration of myofiber nuclear number contributed to increased muscle size in the soleus, but not in plantaris, suggesting that cellular mechanisms regulating muscle size differ between muscles with different fiber type composition.

Activated satellite cells fail to restore myonuclear number in spinal cord transected and exercised rats.

In this study, possible mechanisms underlying soleus muscle atrophy after spinal cord transection and attenuation of atrophy with cycling exercise were studied. Adult female Sprague-Dawley rats were divided into three groups; in two groups the spinal cord was transected by a lesion at T10. One group was transected and killed 10 days later, and another group was transected and exercised for 5 days starting 5 days after transection. The third group served as an uninjured control. All animals received a continuous-release 5'-bromo-2'-deoxyuridine pellet 10 days before they were killed. Transection alone and transection with exercise lead to activation of satellite cells, but only the exercise group showed a trend toward an increase in the number of proliferating satellite cells. In all cases the number of activated satellite cells was significantly higher than the number that divided. Although the number of cells undergoing proliferation increased with exercise, no increase in fusion of satellite cells into muscle fibers was apparent. Spinal cord transection resulted in a 25% decrease in myonuclear number, and exercise was not associated with a restoration of myonuclear number. The number of apoptotic nuclei was increased after transection, and exercise attenuated this increase. However, the decrease in apoptotic nuclei with exercise did not significantly affect myonuclear number. We conclude that apoptotic nuclear loss likely contributes to loss of nuclei during muscle atrophy associated with spinal cord transection and that exercise can maintain muscle mass, at least in the short term, without restoration of myonuclear number.

Early changes in muscle fiber size and gene expression in response to spinal cord transection and exercise.

Muscles of spinal cord-transected rats exhibit severe atrophy and a shift toward a faster phenotype. Exercise can partially prevent these changes. The goal of this study was to investigate early events involved in regulating the muscle response to spinal transection and passive hindlimb exercise. Adult female Sprague-Dawley rats were anesthetized, and a complete spinal cord transection lesion (T10) was created in all rats except controls. Rats were killed 5 or 10 days after transection or they were exercised daily on motor-driven bicycles starting at 5 days after transection and were killed 0.5, 1, or 5 days after the first bout of exercise. Structural and biochemical features of soleus and extensor digitorum longus (EDL) muscles were studied. Atrophy was decreased in all fiber types of soleus and in type 2a and type 2x fibers of EDL after 5 days of exercise. However, exercise did not appear to affect fiber type that was altered within 5 days of spinal cord transection: fibers expressing myosin heavy chain 2x increased in soleus and EDL, and extensive coexpression of myosin heavy chain in soleus was apparent. Activation of satellite cells was observed in both muscles of transected rats regardless of exercise status, evidenced by increased accumulation of MyoD and myogenin. Increased expression was transient, except for MyoD, which remained elevated in soleus. MyoD and myogenin were detected both in myofiber and in satellite cell nuclei in both muscles, but in soleus, MyoD was preferentially expressed in satellite cell nuclei, and in EDL, MyoD was more readily detectable in myofiber nuclei, suggesting that MyoD and myogenin have different functions in different muscles. Exercise did not affect the level or localization of MyoD and myogenin expression. Similarly, Id-1 expression was transiently increased in soleus and EDL upon spinal cord transection, and no effect of exercise was observed. These results indicate that passive exercise can ameliorate muscle atrophy after spinal cord transection and that satellite cell activation may play a role in muscle plasticity in response to spinal cord transection and exercise. Finally, the mechanisms underlying maintenance of muscle mass are likely distinct from those controlling myosin heavy chain expression.

A role for the ETS domain transcription factor PEA3 in myogenic differentiation.

Activation of adult myoblasts called satellite cells during muscle degeneration is an important aspect of muscle regeneration. Satellite cells are believed to be the only myogenic stem cells in adult skeletal muscle and the source of regenerating muscle fibers. Upon activation, satellite cells proliferate, migrate to the site of degeneration, and become competent to fuse and differentiate. We show here that the transcription factor polyomavirus enhancer activator 3 (PEA3) is expressed in adult myoblasts in vitro when they are proliferative and during the early stages of differentiation. Overexpression of PEA3 accelerates differentiation, whereas blocking of PEA3 function delays myoblast fusion. PEA3 activates gene expression following binding to the ets motif most efficiently in conjunction with the transcription factor myocyte enhancer factor 2 (MEF2). In vivo, PEA3 is expressed in satellite cells only after muscle degeneration. Taken together, these results suggest that PEA3 is an important regulator of activated satellite cell function.

Selective accumulation of MyoD and myogenin mRNAs in fast and slow adult skeletal muscle is controlled by innervation and hormones.

Each of the myogenic helix-loop-helix transcription factors (MyoD, Myogenin, Myf-5, and MRF4) is capable of activating muscle-specific gene expression, yet distinct functions have not been ascribed to the individual proteins. We report here that MyoD and Myogenin mRNAs selectively accumulate in hindlimb muscles of the adult rat that differ in contractile properties: MyoD is prevalent in fast twitch and Myogenin in slow twitch muscles. The distribution of MyoD and Myogenin transcripts also differ within a single muscle and correlate with the proportions of fast glycolytic and slow oxidative muscle fibres, respectively. Furthermore, the expression of a transgene consisting of a muscle-specific cis-regulatory region from the myoD gene controlling lacZ was primarily associated with the fast glycolytic fibres. Alteration of the fast/slow fibre type distribution by thyroid hormone treatment or by cross-reinnervation resulted in a corresponding alteration in the MyoD/Myogenin mRNA expression pattern. These findings show that the expression of specific myogenic helix-loop-helix regulators is under the control of innervation and humoral factors and may mediate differential control of contractile protein gene expression in adult muscle.