We examined the role of nitric oxide (NO) in muscle repair and regeneration following repetitive eccentric contractions (ECC). A standardized exercise protocol was used to create eccentric contraction-induced injury to the left tibialis anterior muscle of 48 male Wistar rats (body wt 250-350 g), using a customized isokinetic test device and a bout of 40 ECCs under electrical stimulation. A nitric oxide synthase inhibitor, N(G)-nitro-l-arginine-methyl ester (l-NAME; 35 mg kg-1 day-1), was included in the diet for half the animals (n = 24) beginning 3 days prior to the ECC and continuing throughout the experiment, whereas the other half (n = 24) received a control diet. ECC/+l-NAME and ECC/-l-NAME were killed after the ECC protocol at 0, 1, 3 and 7 days (n = 6 on each day). An unexercised contralateral limb with and without l-NAME infusion served as a respective control muscle at each time point. Muscle NO content, skeletal muscle damage, leukocyte infiltration, calpain activity, and MyoD and myogenin expression were assessed. NO has both pro-inflammatory and anti-inflammatory properties, and several possible roles for NO in skeletal muscle damage have been postulated. NO content was greater in the ECC/-l-NAME group at all time points (p < 0.05) compared to ECC/+l-NAME. Additionally, significant differences in NO content were observed on day 0 (p < 0.05), and day 3 (p < 0.05), ECC/+l-NAME versus ECC/-l-NAME. One day following the bout of ECC, and NO levels were increased in the ECC/-l-NAME group. Three days following ECC, there was greater myofiber damage (measured by β-glucuronidase activity) and leukocyte invasion in the ECC/-l-NAME group as compared to the ECC/+l-NAME group. One day after ECC, calpain activity was significantly increased in ECC/-l-NAME compared with control muscles (p < 0.05). On days 3 and 7, Myo-D and myogenin gene expression was increased in both groups; however, the degree of regeneration was less in the ECC/+l-NAME-treated animals. These data suggest that NO dynamics have important implications in the regulation of various factors during skeletal muscle regeneration following damaging eccentric muscle contractions.
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Acknowledgments The authors thank Dr. Mizuki Sudo (Central Research Institute for Physical Activity, Fukuoka University) for her expert technical assistance. This work was supported by Grant-in-Aid for Scientific Research (C) from the ministry of Education, Culture, Sports, Science and Technology of Japan (21500545). The work presented here was carried out in collaboration between all authors. Sakurai, Kashimura and Best defined the research theme. Sakurai and Izawa designed methods and experiments, carried out the laboratory experiments, analyzed the data, interpreted the results and wrote the paper. Kano and Ohno co-designed the dispersal and colonization experiments, and co-worked on associated data collection and their interpretation. Ji co-designed experiments, discussed analyses, interpretation, and presentation. All authors have contributed to, seen, and approved the manuscript.
- Eccentric muscle contraction
- Nitric oxide
- Skeletal muscle