Early NOX2 activation is crucial in phenylephrine-induced hypertrophy of H9c2 cells

Increased production of reactive oxygen species (ros) is implicated in the process of cardiomyocyte hypertrophy, known to be induced by different stimuli, like angiotensin ii, pressure overload and phenylephrine (pe). Although targeting adrenoreceptors and therefore sharing common signaling processes, recent studies point to different mechanisms activated by these stimuli, in which different nox isoforms, as important sources of ros, might play a role. As such, nox2 was shown to be the main isoform involved in angiotensin ii-induced hypertrophic responses, whereas nox4 was shown to be essential in pressure overload. The role of nox isoforms in pe-induced cardiomyocyte hypertrophy, however, is unknown. Rat neonatal cardiomyoblasts (h9c2 cells) were incubated with 100 μm pe to induce hypertrophy. The effects of pe-induced hypertrophy on the expression of different nox proteins and nox-mediated ros production were studied via electron microscopy and digital-imaging microscopy. Incubation of h9c2 cells with pe significantly induced hypertrophy after 24 and 48 hours. However, at these time points no significant differences in nox/ros expression levels were found. Interestingly, in contrast to nox1 and nox4, nox2 expression increased significantly up to 4 hours after pe stimulation, coinciding with ros production in the cytoplasm as well as the nucleus. Furthermore, inhibition of nox-mediated ros production with apocynin, diphenylene iodonium (dpi) or gp91 docking sequence (gp91ds)-tat peptide during these first 4 hours of pe stimulation significantly inhibited hypertrophy of h9c2 cells, both after 24 and 48 hours of pe stimulation. Early nox2-mediated ros production is crucial in pe-induced hypertrophy of h9c2 cells.