Mechanism of glutathione depletion during simulated ischemia-reperfusion of H9c2 cardiac myocytes

Abstract

It has been reported that myocardial glutathione content is decreased during ischemia-reperfusion, but the mechanism of glutathione depletion has remained unclear. The present study tested whether osmotic stress is involved in the glutathione depletion during ischemia. Six hours of hypoxic acidosis with either high CO₂ tension or low HCO₃⁻ concentration, which simulates the ischemic condition, resulted in a significant decrease of glutathione content and the glutathione depletion was prevented by hyperosmolarity. High-CO₂ acidosis alone without hypoxia induced a similar degree of glutathione depletion. Intracellular pH was lowered by high-CO₂ acidosis to 6.41 ± 0.03 in 15 min. Meanwhile, the cell size gradually increased and reached ∼110% in 10 min and the increased cell size was maintained for at least 30 min, which was also prevented by hyperosmolarity. Subsequent experiments observed the effects of simulated reperfusion on the glutathione content. Measured in 1 h after the hypoxic acidotic reperfusion, the glutathione content was further decreased compared to the level at the end of ischemia, which was not suppressed by increasing the osmolarity of reperfusion solution. The degree of glutathione depletion during hypoxic reperfusion with normal pH was similar to the hypoxic acidotic reperfusion group. On the other hand, normoxic reperfusion was not accompanied by further depletion of glutathione content. Based on these results, it was concluded that ischemia induces the glutathione depletion via osmotic stress, which results from intracellular acidification, and the glutathione content is further decreased during reperfusion through a mechanism other than oxygen toxicity.

Keywords::

• Ischemia
• osmotic stress
• glutathione
• acidosis
• reperfusion

This paper was first published online on Early Online on 14 July 2011.