INTRODUCTION: Nitric oxide (NO) has an impact on skeletal muscle. Previous studies suggest that ischemia/reperfusion (I/R) leads to an imbalance of NO products, thus preventing muscle function recovery. This study evaluated the effects of combining the iNOS inhibitor 1400W and the NO donor SNAC on contractile function and nitric oxide synthase (NOS) mRNA and protein expression in reperfused rat skeletal muscle.

METHODS: 120 rats were divided into groups with reperfusion times of 3 hours, 24 hours, and 7 days after 3 hour ischemia. For each time period, rats were further divided into sham operation, control, 1400W only, and 1400W plus SNAC groups. Muscle contractile function was evaluated as a percentage of contralateral normal muscle function without I/R injury. Real-time PCR and Western blot analysis were used to determine expression of mRNA and protein, respectively.

RESULTS: All three-time periods of reperfusion showed that combination treatment of 1400W and SNAC significantly improved twitch and tetanic muscle force compared to both control and 1400W only. For both the 3 hour and 24 hour groups, sham surgery allowed significantly greater contractile forces than all three other groups with values of recovery ranging from 70-95% for twitch and tetanic force. The 3-hour reperfusion group showed significant improvement in twitch and overall tetanic muscle force by using combination therapy. For the 24-hour group, twitch force was 14.9 percent for control, 25.9 for 1400W only, and 29.6 for 1400W+SNAC. These differences were significant compared to control for both 1400W only (p<0.01) and 1400W+SNAC (p<0.001). Overall tetanic function showed statistical significance between control and 1400W only (p<0.05), 1400W and 1400W+SNAC (p<0.01), and control and 1400W+SNAC (p<0.001). Preliminary data for 7 days also shows improved functional recovery with 1400W+SNAC. After 3 h reperfusion, iNOS mRNA expression was up regulated to 93-fold of normal in controls, but only 19-fold in both sham and 1400W+SNAC groups. There was a significant (p<0.05) decrease in the 1400W+SNAC group. Efforts towards determining expression of muscle mRNA and protein during different reperfusion times are being continued.

CONCLUSIONS: The results demonstrate that combination treatment of 1400W and SNAC promotes functional recovery in reperfused skeletal muscle, in which SNAC supplements the loss of NO from constitutive NOS and 1400W inhibits excessive NO generated from iNOS. This combination recovers NO balance and is more effective than either SNAC or 1400W alone. Our data supports that each distinct NOS plays a different and important role in I/R injury.