Nnos Expression Patterns In The Differentiation Of Primary Myoblasts

The differentiation potential of myoblasts is important for maintaining muscle homeostasis. We recently reported that neuronal nitric oxide synthase (nNOS) protein and gene expressions were decreased during muscle atrophy in cast-immobilized muscle. However, whether nNOS is expressed during myoblast differentiation. In this study, we found that nNOS protein and gene expression increased during primary myoblast differentiation. PURPOSE: To investigate how nNOS expression changes during the differentiation of primary myoblasts. METHODS: Skeletal muscle stem cells were isolated from the gastrocnemius of male C57BL/6 J mice (9 -weeks -old) and cultured. To confirm differentiation of primary myoblasts and nNOS expression, Pax7 (a quiescent muscle stem cell marker) and nNOS immunostaining was performed on muscle stem cells cultured for 4 days (day 4 primary myoblasts: d4 PM) or cultured for 5 days + differentiated for 1 day (day 6 primary myoblasts: d6 PM). The mRNA expression of Nos1μ, the that skeletal muscle- specific nNOS isoform, was examined in d4 PM and d6 PM cells using real-time PCR. To determine nNOS protein expression level and pattern, western blotting was performed. Statistical analyses were performed using SPSS version 27. RESULTS: Immunostaining of primary myoblasts showed that nNOS was expressed in both d4 PM cells and d6 PM cells. Furthermore, Nos1μ gene expression level was significantly higher in d6 PM cells compared with that in d4 PM cells (mean ± SE, 1.00 ± 0.09 vs. 41.15 ± 6.13, P < 0.01). nNOS protein expression level was also significantly higher in d6 PM cells compared with d4 PM cells (1.00 ± 0.13 vs. 1.85 ± 0.2, P < 0.01). CONCLUSIONS: These data suggest that nNOS may be involved in the differentiation of primary myoblasts.