Background: ATP is the major energy source for myotube contraction,and is quickly produced to compensate ATP consumption and to maintain sufficient ATP level.ATP is consumed mainly in cytoplasm and produced in mitochondria during myotube contraction.To understand the mechanism of ATP homeostasis during myotube contraction,it is essential to monitor mitochondrial ATP at single-cell level,and examine how ATP is produced and consumed in mitochondria.Methods: We established C2C12 cell line stably expressing fluorescent probe of mitochondrial ATP,and induced differentiation into myotubes.We gave electric pulse stimulation to the differentiated myotubes,and measured mitochondrial ATP.We constructed mathematical model of mitochondrial ATP at single-cell level,and analyzed kinetic parameters of ATP production and consumption.Results: We performed hierarchical clustering analysis of time course of mitochondrial ATP,which resulted in two clusters.Cluster 1 showed strong transient increase,whereas cluster 2 showed weak transient increase.Mathematical modeling at single-cell level revealed that the ATP production rate of cluster 1 was larger than that of cluster 2,and that both regulatory pathways of ATP production and consumption of cluster 1 were faster than those of cluster 2.Cluster 1 showed larger mitochondrial mass than cluster 2,suggesting that cluster 1 shows the similar property of slow muscle fibers,and cluster 2 shows the similar property of fast muscle fibers.Conclusion: Cluster 1 showed the stronger mitochondrial ATP increase by larger ATP production rate,but not smaller consumption.Cluster 1 might reflect the larger oxidative capacity of slow muscle fiber.