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.

Air-Pollutant-Philic Plants for Air Remediation

In this communication, we review our work over two decades on air-pollutant-philic plants that can grow with air pollutants as the sole nutrient source. We believe that such plants are instrumental in mitigating air pollution. Our target air pollutant has been atmospheric nitrogen dioxide (NO2), and our work on this subject has consisted of three parts: Variation in plants’ abilities to mitigate air pollutants among naturally occurring plants, genetic improvement of plants’ abilities to mitigate air pollutants, and the plant vitalization effect of NO2. So far, an estimation of the half-life of nitrogen derived from NO2 uptake in plants belonging to the 217 taxa studied to date has shown no plants to be naturally occurring air-pollutant-philic. However, we found that an enormous difference exists in plants’ ability to uptake and assimilate atmospheric NO2. Future studies on the causes of this process may provide an important clue to aid the genetic production of plants that are effectively air-pollutant-philic. Both genetic engineering of the genes involved in the primary nitrate metabolism and genetic modification by ion-beam irradiation failed to make plants air-pollutant-philic, but mutants obtained in these studies will prove useful in revealing those genes critical in doing so. During our study on air-pollutant-philic plants, we unexpectedly discovered that prolonged exposure of plants to a sufficient level of NO2 activates the uptake and metabolism of nutrients that fuel plant growth and development. We named this phenomenon “the plant vitalization effect of NO2” (PVEON). Investigations into the mechanisms and genes involved in PVEON will provide an important clue to making plants air-pollutant-philic in the future.