Heat shock-induced attenuation of hydroxyl radical generation and mitochondrial aconitase activity in cardiac H9c2 cells

A mild heat shock (hyperthermia) protects cells from apoptotic and necrotic deaths by inducing overexpression of various heat shock proteins (Hsps). These proteins, in combination with the activation of the nitric oxide synthase (NOS) enzyme, play important roles in the protection of the myocardium against a variety of diseases. In the present work we report that the generation of potent reactive oxygen species (ROS), namely ·OH in cardiac H9c2 cells, is attenuated by heat shock treatment (2 h at 42°C). Western blot analyses showed that heat shock treatment induced overexpression of Hsp70, Hsp60, and Hsp25. The observed ·OH was found to be derived from the superoxide (O₂^–·) generated by the mitochondria. Whereas the manganese superoxide dismutase (MnSOD) activity was increased in the heat-shocked cells, the mitochondrial aconitase activity was reduced. The mechanism of O₂^–· conversion into ·OH in mitochondria is proposed as follows. The O₂^–· leaked from the electron transport chain, oxidatively damages the mitochondrial aconitase, releasing a free Fe²⁺. The aconitase-released Fe²⁺ combines with H₂O₂ to generate ·OH via a Fenton reaction and the oxidized Fe³⁺ recombines with the inactivated enzyme after being reduced to Fe²⁺ by other cellular reductants, turning it over to be active. However, in heat-shocked cells, because of higher MnSOD activity, the excess H₂O₂ causes irreversible damage to the mitochondrial aconitase enzyme, thus inhibiting its activity. In conclusion, we propose that attenuation of ·OH generation after heat shock treatment might play an important role in reducing the myocardial ischemic injury, observed in heat shock-treated animals. proteins; free radicals; spin trapping; reactive oxygen species