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 center dot OH in cardiac H9c2 cells, is attenuated by heat shock treatment (2 h at 42 degrees C). Western blot analyses showed that heat shock treatment induced overexpression of Hsp70, Hsp60, and Hsp25. The observed center dot OH was found to be derived from the superoxide (O(2)(-)center dot) 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 O(2)(-)center dot conversion into center dot OH in mitochondria is proposed as follows. The O(2)(-)center dot leaked from the electron transport chain, oxidatively damages the mitochondrial aconitase, releasing a free Fe(2+). The aconitase-released Fe(2+) combines with H(2)O(2) to generate center dot OH via a Fenton reaction and the oxidized Fe(3+) recombines with the inactivated enzyme after being reduced to Fe(2+) by other cellular reductants, turning it over to be active. However, in heat-shocked cells, because of higher MnSOD activity, the excess H(2)O(2) causes irreversible damage to the mitochondrial aconitase enzyme, thus inhibiting its activity. In conclusion, we propose that attenuation of center dot OH generation after heat shock treatment might play an important role in reducing the myocardial ischemic injury, observed in heat shock-treated animals.