Monitoring and mathematical modeling of mitochondrial ATP in myotubes at single-cell level reveals two distinct population with different kinetics

Backgrounds 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.Conclusions 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.

Influence of morphology of hollow silica-alumina composite spheres on their activity for hydrolytic dehydrogenation of ammonia borane

Hollow silica-alumina composite spheres were prepared by a polystyrene(PS)template method using various amounts of PS suspension.Homogeneous hollow spheres prepared using 40 g were found to be with a diameter of about 300 nm in scanning electron microscopy,and transmission electron microscopy demonstrated their hollow sphere morphology.From the nitrogen adsorption isotherm results,the homogeneous hollow spheres prepared using 40 g of the PS suspension were found to be an ordered pore structure.The activities of the hollow spheres prepared using various amounts of the PS suspension for hydrolytic dehydrogenation of ammonia borane were compared.The results showed that 10,7,and 6 mL of hydrogen were evolved from the aqueous ammonia borane solution in about 40 min in the presence of the hollow spheres prepared using 40,80,and 120 g of PS suspension,respectively.The homogeneous hollow spheres with an ordered pore structure showed the highest activity among all the hollow spheres.The amount of acid sites and the coordination number of aluminum active species were characterized using neutralization titration and solid-state ^(27)Al magic angle spinning nuclear magnetic resonance spectroscopy.The homogeneous hollow spheres with an ordered pore structure had high amount of acid sites and 4-coordinated aluminum species.The relative proportion of 4-coordinated aluminum species was related to the dispersion of aluminum species.These results indicate that the homogeneous hollow spheres with an ordered pore structure showed the high activity because of high amount of acid sites induced by the highly dispersed aluminum species.