GW24-e0440 Diazoxide inhibits the opening of MPTP after hypoxia/reoxygenation injury in myocardial cells through alleviation of calcium overload and ATP deficiency

Objectives

Diazoxide, a mitoK_ATP channel opener can inhibit the opening of mitochondrial permeability transition pore (MPTP) in myocardial cells after hypoxia/reoxygenation injury. The mechanisms are not clear. We hypothesised that the protective effects of Diazoxide might be related to the modulation of the intracellular calcium and ATP homeostasis.

Methods

Cultured neonatal rats cardiomyocytes were subjected to 1 hour hypoxia followed by 2 hours reoxygenation (H/R) and divided randomly into the following groups: (1) control: no H/R and no interventions; (2) H/R; (3) Diaz: 1000 μmol/L diazoxide were given; (4) 5-HD + Diaz: 1000 μmol/L 5-hydroxydecanoic acid (5-HD, a mitoK_ATP channel inhibitor) were given prior to diazoxide; (5) Atr + Diaz: 100 μmol/L atractyloside (Atr, a MPTP opener) were given before diazoxide; (6) Atr: 100 μmol/L atractyloside were given alone; (7) NaOH: 0.01 mmol/L NaOH (a solvent for all those drugs) were given alone. All the drugs were given at the 15^th min after reoxygenation.

Results

After intervention with Diazixide during reoxgenation, the opening of MPTP was decreased significantly (0.96 ± 0.02 in Diaz vs 0.72 ± 0.04 in H/R, p < 0.01), the generation of reactive oxygen species (ROS) was declined (1.16 ± 0.15 in Diaz vs 1.67 ± 0.28 in H/R, p < 0.05), the mitochondrial membrane potential (Δψm) was maintained (0.88 ± 0.07 in Diaz vs 0.75 ± 0.01 in H/R, p < 0.01), the cardiomyocyte apoptosis was reduced (21.93 ± 3.26% in Diaz vs 32.82 ± 3.02% in H/R, p < 0.05), the intracellular calcium overload was controlled (1.29 ± 0.03 in Diaz vs 1.79 ± 0.24 in H/R, p < 0.01) and the intracellular ATP level was restored (34.58 ± 9.67 in Diaz vs 8.47 ± 2.15 in H/R, p < 0.01). Moreover, treated with Diazoxide during reoxygenation, the releasing of cytochrome C (Cyt C) and apoptosis inducing factor (AIF) from mitochondria to cytoplasm was blocked significantly. 5-HD abolished the cardioprotective effects of Diazoxide and atractyloside weakened the effects of Diazoxide.

Conclusions

The cardioprotective effect of mitoK_ATP channel opener is induced by inhibiting the opening of MPTP via alleviation of calcium overload and ATP deficiency so as to prevent mitochondrial dysfunction and cell apoptosis during early reoxygenation.